CN109475615B

CN109475615B - Novel immunogenic formulations comprising linear or branched polyacrylic acid polymer adjuvants

Info

Publication number: CN109475615B
Application number: CN201780044245.6A
Authority: CN
Inventors: G·里加尤特; A·G·A·L·帕里索特; K·德鲁卡; C·M·P·安德莱奥尼; L·莱莫卢; M·加里诺特; J-F·科特; P·普罗伯克-奎莱克特; J·哈恩斯勒; V·查波恩; P·塔拉加
Original assignee: Sanofi Pasteur Inc; Merial Inc
Current assignee: Sanofi Pasteur Inc; Boehringer Ingelheim Animal Health USA Inc
Priority date: 2016-06-17
Filing date: 2017-06-15
Publication date: 2023-04-07
Anticipated expiration: 2037-06-15
Also published as: EP3471760A1; BR112018076015A2; MX2018015787A; JP2019524873A; US11554170B2; AU2017286727B2; AU2017286727A1; BR112018076015A8; JP7018941B2; KR20190044052A; WO2017218819A1; CN109475615A; CA3027877A1; US20170360923A1

Abstract

The present invention provides novel immunization and vaccine formulations comprising a newly applied non-crosslinked polyacrylic acid polymer adjuvant. The adjuvant may be combined with various immunogens in order to produce a safe and effective vaccine when administered to a wide range of target animals. The immunogen may include, but is not limited to: an inactivated pathogen, an attenuated pathogen, a subunit, a recombinant expression vector, a plasmid, or a combination thereof. The animals may include, but are not limited to: humans, murines, canines, felines, equines, porcines, ovines, caprines and bovines.

Description

Novel immunogenic formulations comprising linear or branched polyacrylic acid polymer adjuvants

Cross Reference to Related Applications

U.S. provisional application No.62/351,492, filed 2016, 6, 17, and incorporated herein by reference in its entirety.

Is incorporated by reference

Any of the foregoing applications and all documents cited therein or during prosecution thereof ("application cited documents") and all documents cited or referenced in application cited documents, as well as all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, are hereby incorporated by reference, along with any manufacturer's instructions, descriptions, product specifications, and product specifications for any products mentioned herein or in any document incorporated by reference herein, and may be used in the practice of the present invention. Citation or identification of any such document in this application is not an admission that the document is available as prior art to the present invention and does not reflect any view of the validity, patentability, and/or enforceability of such cited patent document. All sequences referenced herein by GenBank accession numbers are incorporated herein by reference in their entirety and as described in GenBank at the filing date of the present application.

Technical Field

The present invention is in the field of vaccines. In particular, the present invention relates to specific adjuvants and adjuvant compositions, and methods of preparing such adjuvants and adjuvanted compositions.

Background

It has been proposed to use polymers comprising acrylic acid units as adjuvants in vaccine compositionsAnd (3) preparing. In most cases, polyacrylic acid polymers recommended as adjuvants are crosslinked polymers. For example, US3,790,665 and US3,919,411 describe the use of acrylic acid polymers cross-linked with polyallyl sugars as adjuvants. Adjuvants corresponding to crosslinked polymers of acrylic or methacrylic acid are also described in US7,163,926, they are crosslinked in particular with polyalkenyl ethers of sugars or polyols. Such polymers are described in

Under the name of (2). The use of CARBOPOL974P, 934P, and 971P, which are cross-linked polymers with high Mw (i.e., about 300 million for 974P, according to the data provided by the manufacturer), is described in US7,163,926, EP1058558, and WO 2009/118523.

Some research efforts have focused on using linear polyacrylic acid polymers or acrylic acid/acrylate copolymers with low weight:

WO2005/065712 proposes a complex comprising a narrow molecular weight distribution polymer comprising units derived from an acrylic acid or a salt thereof, and a substance having pharmacological activity against a pathogenic organism or cancer or one or more antigens or immunogens. The polymer may be a homopolymer or copolymer of acrylic or methacrylic acid or a salt thereof. The molecular weight is advocated to be 100000 or less.

US6,610,310 and EP0804234 describe the use of polymers having anionic and hydrophobic constitutive repeating monomer units. In particular, EP0804234 discloses the use of polymers composed in part of acrylic acid units (anionic constitutional repeating units) and acrylate ester units (constitutive hydrophobic repeating units) as vaccine adjuvants in aqueous solutions. In these documents polyacrylic acid polymers are used

907 compares to its advocated homologously partially esterified polyacrylic acid polymer and provides a poor immune response. Similarly, the L.Hilgers et al publications Vaccine,2000, 18, 3319-3325 and Vaccine1998 Vol.16, 16, 1575-1581 also disclose such esterification polymerizationsThe use of (b) and teaches that in most cases the use of alkyl esters of polyacrylic acid provides a better immune response. />

907 is a polyacrylic polymer, which is no longer available and whose properties cannot be reliably determined. It belongs to the CARBOPOL family and is called a cross-linked polymer. The polymer has a weight-average molecular weight Mw different from that of the document in that: the publication Vaccine,1998, volume 16, stage 16, pages 1575-1581 provides a weight average molecular weight Mw of 450kDa without accuracy for the method used for its determination. It does not mention its polydispersity index. In contrast, "Liquid Detergents", surfactant series Science, volume 67, page 147 (1996, CRC Press, publishers. About>

907 are questionable. Furthermore, in most cases the Mw data given by the producer differ from the Mw that can be determined by a standardized method, as shown in the examples of the present patent application.

In addition to the uncertainty regarding the precise characteristics of existing polymeric adjuvants, there is a continuing need to develop new adjuvants with improved safety and efficacy properties. The present disclosure meets these needs by providing safe and effective immunization and vaccine formulations comprising non-crosslinked acrylic acid polymer adjuvants.

Summary of The Invention

In a first aspect, the present disclosure provides formulations comprising a novel class of polymers as vaccine adjuvants. As disclosed herein, such polymers have demonstrated safety and efficacy in adjuvant formulations of various antigens, useful for administration to various animal species. Such polymers give advantageous adjuvant properties compared to other families of polyacrylic acid polymers used in the prior art.

In one embodiment of the first aspect, the present invention provides a family of polymers that are particularly effective as adjuvants. In one embodiment, the present invention provides a class of polymers that unexpectedly promote strong Th-1 responses in addition to Th-2 responses.

In addition, some polymers selected according to the present invention produce adjuvant compositions, and thus vaccine compositions. In a particular embodiment, the vaccine composition of the invention is safer, in particular with regard to reproducibility and reduction of contaminants that are generally incompatible with vaccine storage stability. In a more specific embodiment, the selected polymer is also stable and sterilizable by autoclaving.

In this context, the present invention relates to a pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use as an adjuvant in a vaccine composition, characterized in that said polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 350-650 kDa.

In particular, the polyacrylic acid polymer salt is composed entirely of units corresponding to the acrylate salt, or is composed entirely of units corresponding to the free acid form of acrylic acid and units corresponding to the acrylate salt.

Advantageously, the polyacrylic acid polymer salt comprises less than 0.005%, preferably less than 0.001% w/w, of oxidizing agent, based on the total dry weight of the polyacrylic acid polymer salt; and/or comprises less than 0.005%, preferably less than 0.001% w/w of persulfate, based on the total dry weight of the polyacrylic acid polymer salt.

In a more specific embodiment, the polyacrylic acid polymer is reacted with Na ⁺ A salt.

In particular embodiments, the polyacrylic acid polymer salt has a polydispersity index of less than or equal to about 4, preferably less than or equal to about 2.5.

In a specific embodiment, the polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 4; or has a weight-average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 4; or has a weight-average molecular weight Mw in the range from 380 to 620kDa and a polydispersity index of less than or equal to 2.5; or has a weight-average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 2.

Advantageously, the polyacrylic acid polymer salt comprises less than 0.005% w/w of acrylic acid monomers in free acid form or in salt form, based on the total dry weight of the polyacrylic acid polymer salt.

According to an advantageous embodiment, the polyacrylic acid polymer salt is diafiltered and sterilized.

Advantageously, the polyacrylic acid polymer salts described in the present invention are used to enhance Th1 immune responses obtained using vaccine compositions. The Th1 immune response is higher than that obtained when using a lower molecular weight Mw polyacrylic acid polymer salt as adjuvant.

Yet another aspect of the present invention relates to a process for preparing a pharmaceutically acceptable salt of a polyacrylic acid polymer according to the present invention, comprising the sequential steps of:

a) A solution of a polyacrylic acid polymer is obtained,

b) Purifying the solution of polyacrylic acid polymer to eliminate impurities, and

c) The solution of purified polyacrylic acid polymer is sterilized.

The invention also relates to a method for storing the polyacrylic acid polymer solution according to the invention, comprising such a preparation method followed by a storage step of the resulting pharmaceutically acceptable salt of the polyacrylic acid polymer in solution.

In a third aspect, the invention also relates to a vaccine composition comprising at least one vaccine agent (e.g., an immunogen or a nucleic acid encoding an immunogen) and a pharmaceutically acceptable salt of a polyacrylic polymer described herein. In a particular embodiment, the immunogen may be selected from: inactivated pathogens, attenuated pathogens, subunit antigens, purified antigens, unpurified antigens, or antigens recombinantly produced using bacterial, yeast, plant, insect, or animal cells, expression vectors including plasmids, and the like. The antigen may be purified by methods well known in the art, including but not limited to ultrafiltration, ultracentrifugation, size exclusion gel filtration, ion exchange chromatography, and PEG purification. The pathogen may be derived from bacteria, viruses, protozoa or fungi, or the immunogen may constitute an antitoxin.

In another aspect, the invention provides a method of inducing an immune response in a vaccine against a pathogen, the method comprising administering a vaccine composition of the invention for vaccination.

It is noted that in this disclosure, and particularly in the claims, terms such as "comprising," "including," and the like can have the meaning attributed to those terms in U.S. patent law; for example, they can mean "including", "comprising", "containing", etc.; and terms such as "consisting essentially of" and "consisting essentially of have the meaning attributed to them by U.S. patent law, e.g., they allow for elements not expressly referenced, but exclude elements found in the prior art or those that affect the essential features or novelty of the present invention.

As used herein, the term "about" refers to an area approximately at or around the circumference. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower bounds of the numerical values set forth above. Generally, the term "about" is used herein to change a numerical value above and below the stated value by a 10% variation. In one aspect, the term "about" refers to plus or minus 20% of the numerical value of its number of uses. Thus, about 50% means in the range of 45% -55%. The recitation herein of numerical ranges by endpoints includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about".

These and other embodiments are disclosed or are apparent from and encompassed by the following detailed description.

Brief Description of Drawings

A full and enabling disclosure of the present invention, including the best mode thereof, directed to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying figures wherein:

FIG. 1 is a schematic showing the antibody titers (IgG 1 and IgG2 a) in D0, D21 and D35 immunized OF1 mice, each mouse injected with 2.5 μ g PS5-rEPA at a time, either alone or co-injected with 200 μ g CARBOPOL, PAA20 or PAA225000;

FIG. 2 is a schematic diagram showing the geometric mean neutralizing antibody titer (GMT) of sera from a group of C57BL/6 mice immunized with 2 μ g hCMV-gB and squalene emulsion, PAA3000, PAA6000, PAA50000, PAA60000, PAA20, or PAA225000 as determined for MRC5 fibroblasts;

FIG. 3 is a schematic showing GMT of the group of FIG. 2 as determined by serum neutralization of ARPE-19 cells (human epithelial cells);

figure 4 is a schematic showing serum IgG1 antibodies to hCMV-gB antigen for the panel of figure 2, as determined by ELISA;

figure 5 is a schematic showing serum IgG2c antibodies to hCMV-gB antigen for the panel of figure 2, as determined by ELISA;

FIG. 6 is a schematic diagram showing IL5 cytokine levels of the panel of FIG. 2 as determined using the CBA agile combination kit;

figure 7 is a schematic diagram showing IFN γ cytokine levels of the set of figure 2 as determined using the CBA flexible combination kit;

FIG. 8 is a graph showing the treatment of rabies in inactivated + PAA225000; AF03; PAA60000; or a representation of rabies serology of a squalene emulsion vaccinated group of canines;

FIG. 9 is a drawing showing recombinant influenza + (1) PBS mediated by canarypox; (2) CARBOMER (4 mg/ml); (3) PAA60000 (4 mg/ml); or (4) PAA225000 (4 mg/ml) schematic representation of CIV serology for the vaccinated canine group. Group 5 received PBS only (i.e., neither recombinant influenza antigen nor adjuvant);

FIG. 10 is a schematic showing an expansion of day 41 serological data shown in FIG. 9;

figure 11 is a schematic showing influenza antibody titers determined for the mean SRH of groups of equids vaccinated with vCP1533+ vCP2242 (each containing the HA gene from influenza virus) and one of the following tetanus toxins: (A) CARBOMER (4 mg/mL); (B) PAA60000 (4 mg/mL); (C) PAA225000 (4 mg/mL); (D) ADVAX1 (20 mg/mL); (E) ADVAX2 (20 mg/mL). Group (F) received PBS only (i.e., neither antigen nor adjuvant);

figure 12 is a schematic showing tetanus serology for each equine group to D35. Group (2): the same as described in fig. 11;

FIG. 13 is a schematic showing serological results for tetanus in equine groups generated to D63;

fig. 14 is a schematic showing the mean SpaA serology (to D59) for each porcine group. Group (2): (G1) SpaA + TS6; (G2) SpaA + PAA60000; (G3) SpaA + PAA225000; spaA-FlaB-His + PBS; (G5) SpaA-FlaB-His + PAA225000; (G6) PBS.

Detailed Description

Other objects, features and aspects of the present invention are disclosed or will become apparent in the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied in the exemplary construction. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment. It is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. All references, published patents and patent applications cited in this application are hereby incorporated by reference in their entirety.

Characteristics of polyacrylic acid polymers

The polymer used in the present invention is a linear or branched polyacrylic acid polymer, but it is not a crosslinked polymer. By "polyacrylic acid polymer" we mean a polymer consisting entirely of acrylic acid units. Thus, in the form of a salt, the polyacrylic acid polymer salt is composed entirely of units corresponding to the acrylate salt, or is composed entirely of units corresponding to the free acid form of acrylic acid and units corresponding to the acrylate salt.

The linear or branched polyacrylic acid polymer is obtained by polymerization of only acrylic acid as a monomer. In most cases, polymerization is carried out by radical polymerization using an oxidizing agent as an initiator or a catalyst. The most common oxidizing agents are persulfates (peroxodisulfates), such as sodium or potassium persulfate. Branched polyacrylic acid polymers are described, for example, in Macromolecules 2011, 44, 5928-5936. When the polymer of the present invention is linear, its Mark Houwink slope is greater than or equal to 0.7 (Yan J.K., pei J.J., ma H.L., wang Z.B.2015.Effects of adsorbed on molecular properties, structure, chain formation and degradation kinetics of carboxylic acid course. Carb.polymers.121, 64-70).

"pharmaceutically acceptable salts" of polyacrylic acid polymers refers to salts of the anionic form of the polymer with cations, particularly with pharmaceutically acceptable monovalent cations. Examples of monovalent cations are alkali metal cations, e.g. Na ⁺ Or K ⁺ Or ammonium cations, e.g. NH ₄ ⁺ . In aqueous solutions having a pH of 5.5 to 8, e.g. close to 7, the acidic groups of the polyacrylic acid polymer will be in anionic form, forming salts with cations also present in the aqueous solution. In the polymer we can have acrylic acid units in which the acid groups are in free acidic form, while the other units having acid groups are in salt-forming anionic form. Depending on the pH, the acid groups of the polymer may be completely in the free acid form or, in the case of salts, the acid groups of the polymer may be completely in the form of salts, or some of the acid groups may be in the acidic form while others may be in the form of salts. The preferred salt of polyacrylic acid polymer of the present invention is with Na ⁺ A salt. Thus, whether or not in the embodiments described herein, the polyacrylic acid polymer is preferably in the form of a sodium salt, and in such cases, all of the characteristics (Mw, IP, monomer and persulfate content.) will relate to the salt (i.e., sodium salt) of the polyacrylic acid polymer.

In the specification of the present patent application, the "pharmaceutically acceptable salt of polyacrylic acid polymer" is simply referred to as "polyacrylic acid polymer salt", and preferably polyacrylic acid polymer sodium salt.

The polyacrylic acid polymer salt may be in solid form (precipitate or powder) or preferably in the form of a liquid formulation. The liquid formulation comprises a polyacrylic acid polymer salt and an aqueous solution. Preferably, such formulations have a pH in the range of 5.5 to 8.0. The pH can be obtained by adding a base such as NaOH to the aqueous solution. The aqueous solution may be a buffered aqueous solution obtained using a buffer such as phosphate buffer, TRIS (2-amino-2-hydroxymethyl-1, 3-propanediol), hepes (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), histidine or citrate buffer. The liquid formulation may also contain one or several additional salts, such as NaCl.

According to the invention, it is proposed to use as adjuvant a polyacrylic acid polymer salt or a liquid formulation of a polyacrylic acid polymer salt having one of the following features and any combination of these features, or even all of the following features, as long as they are not mutually exclusive:

-the polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 350-650 kDa;

-the polyacrylic acid polymer salt or the liquid formulation of the polyacrylic acid polymer salt comprises less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, of oxidizing agent; and/or less than 0.005%, preferably less than 0.001% w/w of a persulfate salt, based on the total dry weight of the polyacrylic acid polymer salt;

-the polyacrylic acid polymer salt has a polydispersity index of less than or equal to 4, preferably less than or equal to 2.5;

-the polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 4 or has a weight average molecular weight Mw in the range of 400 to 600kDa and a polydispersity index of less than or equal to 4;

-the polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 2.5 or has a weight average molecular weight Mw in the range of 400 to 600kDa and a polydispersity index of less than or equal to 2;

-the polyacrylic acid polymer salt has a mark-houwink slope greater than or equal to 0.7;

-the polyacrylic acid polymer salt or the liquid formulation of the polyacrylic acid polymer salt comprises less than 0.005% w/w of acid monomers in free acrylic acid form or in salt form, based on the total dry weight of the polyacrylic acid polymer salt.

In particular, the use of polyacrylic acid polymer salts or liquid formulations of polyacrylic acid polymer salts is proposed as adjuvants, characterized in that:

-a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 4, a persulfate content in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, and a content of acrylic acid monomer in free acid form or in salt form in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005% w/w, based on the total dry weight of the polyacrylic acid polymer salt; advantageously, the polyacrylic acid polymer salt has a Mark-Houwink slope of greater than or equal to 0.7, or

-a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 2.5, a persulfate content in the polyacrylic acid polymer salt or in the liquid formulation of the polyacrylic acid polymer salt of less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, and a content of acrylic acid monomers in free acid form or in salt form in the polyacrylic acid polymer salt or in the liquid formulation of the polyacrylic acid polymer salt of less than 0.005% w/w, based on the total dry weight of the polyacrylic acid polymer salt; advantageously, the polyacrylic acid polymer salt has a Mark-Houwink slope of greater than or equal to 0.7, or

-a weight average molecular weight Mw in the range of 400 to 600kDa and a polydispersity index of less than or equal to 4, a persulfate content in the polyacrylic acid polymer salt or in the liquid formulation of the polyacrylic acid polymer salt of less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, and a content of acrylic acid monomers in free acid form or in salt form in the polyacrylic acid polymer salt or in the liquid formulation of the polyacrylic acid polymer salt of less than 0.005% w/w, based on the total dry weight of the polyacrylic acid polymer salt; advantageously, the polyacrylic acid polymer salt has a Mark-Houwink slope of greater than or equal to 0.7, or

-a weight average molecular weight Mw in the range of 400 to 600kDa and a polydispersity index of less than or equal to 2, a persulfate content in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, and a content of acrylic acid monomer in free acid form or in salt form in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005% w/w, based on the total dry weight of the polyacrylic acid polymer salt; advantageously, such polyacrylic acid polymer salts have a mark-houwink slope greater than or equal to 0.7.

Advantageously, the polyacrylic acid polymer salt in the liquid formulation is diafiltered.

Advantageously, the polyacrylic acid polymer salt or the liquid formulation of the polyacrylic acid polymer salt is sterilized. When the polyacrylic acid polymer salt or the liquid formulation of the polyacrylic acid polymer salt is diafiltered, sterilization is performed after diafiltration.

According to the invention, the weight-average molecular weight Mw is obtained by size exclusion chromatography. Advantageously, three detectors will be used after the size exclusion chromatography column: a right angle light scattering detector, a refractive index detector and a four-capillary differential viscometer. The detailed procedures provided in the examples are preferably used in accordance with the invention for determining Mw, IP (polydispersity index), polymer concentration and mark-houwink slope. The dn/dc used to determine Mw is preferably determined using a refractive index detector and a set of polyacrylic acid polymers of known concentration.

The persulfate content and the free acid form or salt form of the acrylic monomer content can be determined by high performance anion exchange chromatography and conductivity detection. Preferably, the protocol detailed in the examples can be used, in particular in paragraph B of the "determination of persulfate and acrylate monomers" section i.2.

Preparation and storage of polyacrylic acid polymers

In the polyacrylic acid polymer base stock, there is a residual monomer content, which corresponds to the unpolymerized acrylic acid or acrylate content. In the polymerization of polyacrylic acid polymers, a polymerization initiator, in most cases an oxidizing agent such as persulfate, is used as a catalyst for initiating polymerization. In polyacrylic acid polymer feedstocks, it is possible to retain residual levels of polymerization initiator (in most cases oxidants, such as persulfates) which are not consumed by the polymerization process.

Polyacrylic acid polymers on the market often lack specifications regarding residual monomer and oxidant content as well as their precise Mw and oligomer content.

According to a preferred embodiment of the present invention, it is proposed to systematically purify the polyacrylic acid polymer raw material purchased for the preparation of adjuvant compositions, in order to avoid the risk of having residual contents of these compounds which may be harmful, in view of the stability of adjuvant-containing adjuvant compositions and vaccine compositions and/or the toxicity of vaccine compositions. In addition, according to the present invention, it has been determined that significant levels of oxidizing agents, such as persulfates, are detrimental to the stability of the polymer under heat treatment and prohibit sterilization by autoclaving.

The present invention relates to a process for the preparation of a pharmaceutically acceptable salt of a polyacrylic acid polymer, in particular a pharmaceutically acceptable salt of a polyacrylic acid polymer as defined in the paragraph "characteristics of polyacrylic acid polymers", comprising the following successive steps:

a) A solution of a polyacrylic acid polymer is obtained,

c) The solution of purified polyacrylic acid polymer is sterilized.

In steps a), b) and c), the solution may be a solution of a polyacrylic acid polymer, which is present directly in the form of the desired pharmaceutically acceptable salt or at least partly in its free acid form. If in step a) the solution is a solution of the polyacrylic acid polymer in free acid form, salification can be performed after purification of step b) and the solution of the desired pharmaceutically acceptable salt is subjected to sterilization of step c). It is also possible to subject the solution of polyacrylic acid polymer in free acid form to sterilization in step c) and to salt formation after sterilization.

At any stage, if salt formation is desired, it can be achieved by introducing a base such as NaOH or KOH in solution, depending on the desired salt.

For example, a solution of a pharmaceutically acceptable salt of a polyacrylic acid polymer is purified and/or sterilized. The solution is, for example, an aqueous buffer solution, in particular using a phosphate buffer or using a TRIS, hepes, histidine or citrate buffer. The aqueous solution of a pharmaceutically acceptable salt of the polyacrylic acid polymer may also contain one or several additional salts, such as NaCl. In this case, according to the process of the present invention, for the preparation of pharmaceutically acceptable salts of polyacrylic acid polymers, in particular pharmaceutically acceptable salts of polyacrylic acid polymers as defined in the paragraph "characteristics of polyacrylic acid polymers", the process comprises the following successive steps:

a) Obtaining a solution of a pharmaceutically acceptable salt of the selected polyacrylic acid polymer,

b) Purifying the solution of the polyacrylic acid polymer salt to eliminate impurities, and

c) The resulting solution of purified polyacrylic acid polymer salt is sterilized.

Advantageously, the polyacrylic acid polymer of the solution of step a) has a Mark-Houwink slope greater than or equal to 0.7. When in solution, the polyacrylic acid polymer is in the form of a salt, and this Mark-Houwink slope relates to the polyacrylic acid polymer salt.

Purification will remove small molecules. Purification may be by dialysis, diafiltration, ultrafiltration or size exclusion chromatography. Diafiltration and ultrafiltration use cross-flow filtration on a porous membrane (also known as tangential flow filtration). The solution containing polyacrylic acid polymer is circulated over the membrane: a portion of the solution comprising small molecules is eliminated in the permeate that will pass through the membrane. Another part of the solution, called the retentate, which contains the purified polyacrylic acid polymer, will be circulated over the surface of the membrane. The retentate can be circulated in a circulation loop and diafiltered or ultrafiltered several times. A solvent (typically an aqueous buffer or saline solution) is added to the retentate and circulated at the same rate as the permeate flow rate to replace the permeate volume so that the volume of the retentate remains constant. The size of the eliminated molecules is determined by the cut-off value (cut-off) of the membrane. Advantageously, membranes with a cut-off value of 1-80kDa, preferably 2-50kDa, can be used. Such membranes are available, for example, in Merck Millipore. The cut-off of the membrane is assessed according to its Nominal Molecular Weight Limit (NMWL) or its molecular weight cut-off (MWCO). For example, a UF membrane rated at 30kD will exclude test proteins with a molecular weight of 30 kilodaltons. Ninety percent of the test protein will remain in the retentate and 10% will pass through to the permeate, resulting in protein concentration if no buffer or saline solution is added to the retentate during the process.

Typically, the flow rate of the retentate cycle is 50-80L/H/m ² . The transmembrane pressure (TMP) is, for example, 0.9+/-0.1bar.

Thus, the purification in step b) may be performed by dialysis, diafiltration, ultrafiltration or size exclusion chromatography. Purification can be carried out on solutions containing from 2 to 50mg/ml, preferably from 10 to 30mg/ml, of polyacrylic acid polymer. When the polymer is in the form of a salt in solution, the concentration relates to the polymer salt.

Advantageously, the purification is carried out by diafiltration using a membrane with a cut-off of 1-80kDa, preferably 2-50 kDa.

Preferably, the purification is carried out under conditions that allow recovery of the polyacrylic acid polymer in a solution having:

-less than 0.005%, preferably less than 0.001% > -w/w of persulfate, based on the total dry matter of the solution obtained after purification (in particular by diafiltration), or more generally less than 0.005%, preferably less than 0.001% > -w/w of oxidizing agent, based on the total dry matter of the solution obtained after purification (in particular by diafiltration), and/or

-less than 0.005% w/w of acrylic acid monomers in free acid or salt form, based on the total dry matter of the solution obtained after purification, in particular by diafiltration.

The weight average molecular weight Mw of the recovered polyacrylic acid polymer may be in the range of 350-650kDa and its polydispersity index is less than or equal to 4.

The purification device is selected to eliminate the desired impurities. For example, when using ultrafiltration or diafiltration for purification, the cut-off of the membrane will be selected according to the impurities to be eliminated. The retention is at least 20kDa and essentially small molecules such as persulfates and monomers are eliminated by cross-filtration. Above a cut-off of 20kDa, larger molecules such as oligomers are also eliminated, as a result of which purification leads to a decrease in IP and an increase in Mw.

Preferably, the diafiltration or ultrafiltration is carried out using the cut-off value of the membrane used, or more generally the purification is carried out under conditions allowing the recovery of the polyacrylic acid polymer in a solution having: a weight average molecular weight Mw in the range from 380 to 620kDa and a polydispersity index of less than or equal to 2.5; or a weight average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 2, and

-less than 0.005%, preferably less than 0.001% w/w of persulfate based on total dry matter of the solution obtained after purification (in particular by diafiltration), or more generally less than 0.005%, preferably less than 0.001% w/w of oxidizer based on total dry matter of the solution obtained after purification (in particular by diafiltration), and/or

Advantageously, the solution obtained after the purification step contains 2-50mg/mL of the polyacrylic acid polymer salt, in particular at least 10 mg/mL.

The main result of the purification process is the elimination of small molecules such as oxidants (i.e., persulfates) and acrylate monomers.

By systematically performing such purification steps, the characteristics of the polymer composition used as an adjuvant can be better determined, and the composition is safer and more stable. The content of acrylic acid monomers suspected of being embryotoxic and teratogenic was significantly reduced.

As mentioned above, depending on the use of the device, in particular the cut-off value of the membrane, diafiltration, dialysis, ultrafiltration or size exclusion chromatography also leads to an increase in the weight average molecular weight of the resulting polyacrylic acid polymer and a decrease in its polydispersity Index (IP). In fact, depending on the technique used, and in particular on the cut-off value of the membrane used, the osmotic characteristics of the gels used in diafiltration, dialysis and ultrafiltration or in size exclusion chromatography, oligomers are also eliminated, and therefore the Mw will increase and the IP will also decrease. In these cases, the composition of the polymer salt is even more controlled.

The sterilization may be performed by sterile filtration or preferably by autoclaving. Sterile filtration was performed on a 0.2 μm pore membrane. The elimination of the oxidizing agent allows sterilization using the pharmacopoeia recommended autoclaving. Autoclaving can be carried out at temperatures of from 100 to 150 ℃ and over a period of from 5 minutes to 1 hour. By the purification step, the resulting polymer is more stable over time and more resistant to heat treatment.

The invention also relates to a method for storing a solution of a pharmaceutically acceptable salt of a polyacrylic acid polymer, in particular a pharmaceutically acceptable salt of a polyacrylic acid polymer as defined in the paragraph "characteristics of a polyacrylic acid polymer", comprising a preparation method as defined according to the invention, followed by a storage step of the pharmaceutically acceptable salt of the polyacrylic acid polymer in the form of the resulting solution. The storage step may last from 1 day to 2 years. In most cases, the storage temperature is in the range of 0 to 30 ℃, in particular 2-8 ℃ or room temperature, usually about 22 ℃. Storage can be carried out directly after the sterilization step c).

Storage of such adjuvants in liquid form is very advantageous and avoids additional handling compared to storage in dry form where polymer re-suspension/dilution is necessary to prepare the vaccine composition.

In particular, when the liquid solution of the polyacrylic acid polymer salt comprises less than 0.005%, preferably less than 0.001% w/w, of oxidizing agent, based on the total dry weight of the polyacrylic acid polymer salt, and/or less than 0.005%, preferably less than 0.001% w/w, of persulfate salt, based on the total dry weight of the polyacrylic acid polymer salt, the solution is particularly stable.

The storage step is performed by placing a solution of the polyacrylic acid polymer salt in a container and storing. During storage, the stored solution contains, for example, 2-50mg/mL of polyacrylic acid polymer salt. A dilution or concentration step may be performed to obtain the desired concentration, for example after step b) of the preparation process. During storage, the polyacrylic acid polymer salt may be, for example, in the form of an aqueous solution or a buffered aqueous solution. The pH of the stored solution is typically between 5.5 and 8, and more preferably between 6.5 and 7.5 (e.g., about 7). A stable pH may be maintained by using a buffer such as Tris buffer, citrate buffer, phosphate buffer, hepes buffer or histidine buffer.

The aqueous solution may also contain one or several additional salts, such as NaCl.

Storage may be carried out by keeping a solution of the polyacrylic acid polymer salt protected from light. For this purpose, dark or opaque containers can be used.

Use of polyacrylic acid polymers as adjuvants

The invention also relates to a polyacrylic acid polymer salt as defined herein, whether or not the embodiments relate to the above paragraph "characteristics of polyacrylic acid polymers", for use as an adjuvant in a vaccine composition or in a vaccine agent for eliciting an immune response in an individual, in particular in a human.

An adjuvant composition for a vaccine comprising an aqueous solution of a pharmaceutically acceptable salt of a polyacrylic acid polymer as defined herein, whether or not the embodiments relate to the "characteristics of polyacrylic acid polymer" in the above paragraph, is also an object of the present invention.

As used herein, "adjuvant" refers to a compound that modulates the immunogenicity of a vaccine composition. Vaccine compositions typically include a vaccine agent, which may be an antigen or a vector encoding an antigen (a live recombinant viral vector or a nucleic acid). More specifically, adjuvants modulate the immunogenicity of antigens presented or encoded by nucleic acids present in the composition. "modulating immunogenicity" includes enhancing the magnitude and/or duration of the immune response induced by the antigen, and in particular includes enhancing the antibody response (especially virus neutralizing or bactericidal antibodies) and/or the cellular immune response (enhancement of CD4+ and/or CD8+ T cell responses).

The polymers selected according to the invention have different advantages, as shown in the examples. For example, they result in an enhanced immune response compared to similar linear or branched polymers of lower Mw.

CD4+ lymphocytes, also known as "helper" T cells, are mediators of immune responses. Traditionally, two types of effector CD4+ T helper cell responses, termed Th1 and Th2, are characterized by cytokine profiles and antibody subtyping. In addition to the Th-2 response normally induced by the human adjuvants of the prior art (aluminium salts, oil-in-water emulsions, results obtained for IL-4, IL-5 and IgG1 antibodies in mice), the use of polyacrylic acid polymers as defined in the present invention has the advantage of promoting a strong Th-1 response (results obtained for IFN-. Gamma., TNF-. Alpha.and IgG2a antibodies in mice). The induction of strong Th-1 immunity is important against viral and intracellular bacterial infections as well as cancer, because the Th-1 immune response supports the activation of macrophages and other killer cells (e.g., CD8+ T lymphocytes or cytotoxic T lymphocytes), thereby killing intracellular pathogens, infected cells, and tumor cells.

The polyacrylic acid polymer salts of the present invention and the vaccine agent may be formulated in the same composition, particularly in an aqueous composition, or in two different compositions and mixed immediately prior to administration.

Vaccine compositions may also be obtained in the form of a kit of parts. The vaccine composition may comprise two vials: one containing the vaccine agent and the other containing the polyacrylic acid polymer salt. In particular, the polyacrylic acid polymer salt in the liquid formulation is contained in a first vial and the vaccine agent, in freeze-dried or lyophilized form, in particular the selected antigen in freeze-dried or lyophilized form, is contained in a second vial. The formulation of polyacrylic acid polymer salts will be used to rehydrate vaccine agents, particularly selected antigens.

The invention also relates to polyacrylic acid polymer salts as defined herein, whether or not said embodiments relate to the "characteristics of polyacrylic acid polymers" of the above paragraphs, for use as adjuvants in vaccine compositions, which enhance the obtained Th1 immune response and/or balance the obtained Th1 and Th2 immune responses. In particular, the polyacrylic acid polymer salts as defined in the present invention are used as adjuvants for vaccine agents for enhancing the immune response of an individual, in particular a human, and for enhancing the obtained Th1 immune response and/or balancing the obtained Th1 and Th2 immune responses.

Vaccine compositions and vaccine agents

The vaccine composition of the invention may comprise any vaccine agent useful in vaccines, such as antigens or vectors encoding antigens (live viral vectors or nucleic acids, including DNA and RNA).

For the purposes of the present invention, the term "antigen" is intended to mean any molecule containing one or more epitopes (linear, conformational or both) which elicits an immune response. Antigens useful in the vaccine compositions of the invention may be live, attenuated, killed, inactivated or non-infectious whole microorganisms, extracts or fragments of microorganisms, subunit forms of natural antigens, recombinant forms or hybrid forms. When it is in subunit form, the nature of the antigen is of little importance. The antigen may be a peptide, protein, glycoprotein, polysaccharide, glycolipid, lipoprotein, lipopeptide, VLP (virus-like particle).

The vaccine agent present in the composition is an antigen or a vector encoding an antigen (recombinant virus or nucleic acid) for use in or for the treatment or prevention of various diseases which may affect humans or animals other than humans, including in particular: diphtheria, tetanus, poliomyelitis, rabies, pertussis, hepatitis a, hepatitis B, hepatitis c, yellow fever, typhoid, varicella, measles, mumps, rubella, japanese encephalitis, influenza, meningitis, cholera, rotavirus, norovirus, rhinovirus, respiratory syncytial virus, herpes simplex virus, papilloma virus, cytomegalovirus, west nile virus, dengue virus, chikungunya virus, infection by HIV (AIDS), streptococcus (streptococci), trachoma and Chlamydia pneumoniae (Chlamydia and pnemoniae), neisseria gonorrhoeae and Neisseria meningitidis (Neisseria gonorrhoeae and menningitis), moraxella (Moraxella), staphylococcus aureus (phus aureus), or listeria B, or Haemophilus influenzae type B (bacillosis), diseases such as bacterial diseases of the species salmonella, e.g., tuberculosis, bacterial diseases, such as bacterial diseases, e.

The antigen may be of bacterial, viral or parasitic nature. Among the antigens suitable for the subject of the invention, mention may be made of: bacterial antigens derived from Clostridium tetani (Clostridium tetani), clostridium diphtheriae (Clostridium diphtheria), bordetella pertussis (Bordetella pertussis), haemophilus influenzae type B (Haemophilus influenzae type B), streptococcus pneumoniae (Streptococcus pneumoniae), neisseria meningitidis (Neisseria meningitidis), shigella sp, salmonella typhi (Salmonella typhi), staphylococcus aureus or Staphylococcus epidermidis (Staphylococcus aureus), mycobacterium tuberculosis (Mycobacterium tuberculosis), chlamydia trachomatis and Chlamydia pneumoniae (Chlamydia and pneumoniae) or Streptococcus sp; a viral antigen derived from hepatitis a, b or c virus, influenza virus, rhinovirus, respiratory syncytial virus, west nile virus, rabies virus, poliovirus, HIV virus, dengue virus, japanese encephalitis virus, yellow fever virus, cytomegalovirus or herpes virus; parasite antigens, in particular from the genus Plasmodium (Plasmodium sp.), leishmania (leishmania sp.) or schistosoma (schistosomas sp.); and a tumor antigen. These antigens may be obtained using genetic recombination methods or using extraction methods well known to those skilled in the art.

In particular, the vaccine agent present in the composition is an antigen or a vector (recombinant virus or nucleic acid) encoding an antigen, derived from staphylococcus aureus or from cytomegalovirus.

The vaccine composition of the invention may be a composition intended for immunization against a single pathogen or cancer, that is to say that it comprises one or more vaccine agents, in particular one or more antigens, of a single pathogen or cancer, or may be a composition intended for immunization against several pathogens or cancers.

The vaccine composition of the invention may also comprise one or several specific vaccine agents, in particular one or several antigens of a single disease, but belonging to different classes of the disease (multiple serotypes or strains or clades, depending on the nature of the agent).

The polyacrylic acid polymer salts and vaccine agents of the present invention may be formulated into compositions with any pharmaceutically acceptable vehicle. In the context of the present invention, the expression "pharmaceutically acceptable vehicle" means a vehicle which is physiologically acceptable for administration to mammals, in particular to humans, while retaining the physiological activity of the composition of the invention, i.e. its ability to induce an immune response. An exemplary pharmaceutically acceptable vehicle is physiological saline buffer. Other physiologically acceptable vehicles are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (18 th edition), ed.A. Gennaro,1990, mack Publishing company, easton, pa.

The pH of the composition is typically between 5.5 and 8, and more preferably between 6.5 and 7.5 (e.g., about 7). A stable pH may be maintained by using a buffer such as Tris buffer, citrate buffer, phosphate buffer, hepes buffer or histidine buffer. Thus, the composition typically comprises a buffer. The composition may be sterile and/or pyrogen-free. The composition may be isotonic with respect to humans.

The composition may also comprise one or several additional salts, such as NaCl.

The compositions of the present invention comprise an immunologically effective amount of a vaccine agent. An "immunologically effective amount" is an amount that, when administered to a subject, is effective to elicit an immune response against the antigen used or produced upon expression of the vector and/or nucleic acid. This amount may vary depending on the health and physical condition of the subject, their age, the ability of the subject's immune system to produce antibodies, the degree of protection desired, the formulation of the vaccine, and the assessment of the medical condition of the treating physician.

The vaccine composition of the present invention may also comprise an allergen, in particular an allergen for desensitization in allergy treatment.

The vaccine composition of the present invention may be administered by any route commonly used for the administration of vaccines. A regimen that results in the induction of the desired immune response will be used. Typically, immunization programs include several authorities. The amount of the composition administered is sufficient to generate the desired immune response.

Preferably, the vaccine composition is in liquid form in view of the good stability of the polyacrylic acid polymer, thereby allowing the use of liquid forms which are less expensive to produce.

Parenteral injection (intramuscular, subcutaneous, intradermal and intravenous) is also preferred. Polyacrylic acid polymers do not cause locally significant side effects after intradermal injection. This may be superior to most other adjuvants (including aluminium salts), which sometimes appear to respond via the intradermal route.

Preferred vaccine compositions of the invention are as follows:

the vaccine composition of the invention comprises at least one vaccine agent and a pharmaceutically acceptable salt of a polyacrylic acid polymer having a weight average molecular weight Mw in the range of 350-650 kDa.

Advantageously, the vaccine composition of the invention comprises 0.1-8mg, preferably 0.1-4mg, and more preferably 0.1-2mg of a pharmaceutically acceptable salt of a polyacrylic acid polymer per dose.

Preferably, the at least one vaccine agent is an antigen or a vector (viral vector or nucleic acid) encoding an antigen derived from a bacterial antigen of the genera clostridium tetani, clostridium diphtheriae, bordetella pertussis, haemophilus influenzae type B, streptococcus pneumoniae, neisseria meningitidis, shigella, salmonella typhi, staphylococcus aureus or staphylococcus epidermidis, mycobacterium tuberculosis, chlamydia trachomatis or chlamydia pneumoniae or streptococcus; viral antigens derived from hepatitis a, b or c virus, influenza virus, rhinovirus, respiratory syncytial virus, west nile virus, rabies virus, poliovirus, HIV virus, dengue virus, japanese encephalitis virus, yellow fever virus, cytomegalovirus or herpes virus; in particular parasite antigens derived from the genus Plasmodium, leishmania or Hematophaga, or tumour antigens. In particular, the vaccine agent present in the composition is an antigen or a vector (recombinant virus or nucleic acid) encoding an antigen, derived from staphylococcus aureus or from cytomegalovirus.

In a preferred embodiment, the vaccine composition of the invention is in liquid form, having a pH in the range of 5.5-8.0. For example, they include phosphate buffer or TRIS, hepes, histidine or citrate buffer.

The polyacrylic acid polymer salt present in the vaccine composition has one of the following characteristics, any combination of such characteristics or even all of the following characteristics, provided they are not mutually exclusive:

-the polyacrylic acid polymer salt or the liquid formulation of the polyacrylic acid polymer salt comprises less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, of oxidizing agent, and/or less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, of persulfate;

-the polyacrylic acid polymer salt or the liquid formulation of polyacrylic acid polymer salt comprises less than 0.005% w/w of acrylic acid monomers in free acid form or in salt form, based on the total dry weight of the polyacrylic acid polymer salt,

-diafiltration and/or sterilisation of a pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer.

In particular, the polyacrylic acid polymer salt present in the vaccine composition is characterized in that:

-a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 4, a persulfate content in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, and a content of acrylic acid monomer in free acid form or in salt form in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005% w/w, based on the total dry weight of the polyacrylic acid polymer salt; advantageously, such polyacrylic acid polymer salts have a Mark-Houwink slope of greater than or equal to 0.7, or

-a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 2.5, a persulfate content in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, and a content of acrylic acid monomers in free acid form or in salt form in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005% w/w, based on the total dry weight of the polyacrylic acid polymer salt; advantageously, the polyacrylic acid polymer salt has a Mark-Houwink slope of greater than or equal to 0.7, or

-a weight average molecular weight Mw in the range of 400 to 600kDa and a polydispersity index of less than or equal to 4, a persulfate content in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005%, preferably less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer salt, and a content of acrylic acid monomer in free acid form or in salt form in a polyacrylic acid polymer salt or in a liquid formulation of a polyacrylic acid polymer salt of less than 0.005% w/w, based on the total dry weight of the polyacrylic acid polymer salt; advantageously, the polyacrylic acid polymer salt has a Mark-Houwink slope of greater than or equal to 0.7, or

The invention also relates to a vaccine composition of the invention for use in generating an immune response in an individual, in particular a human. The subject of the present invention is also a method for increasing the immune response in an individual, in particular a human, comprising the step of administering to the individual in need thereof an immunologically effective amount of a composition according to the invention. The subject may be a human or an animal selected from canine, feline, bovine, porcine, equine or ovine species, and ferret and avian species.

The invention also relates to a vaccine composition of the invention for generating an immune response in an individual, in particular in a human, while enhancing the obtained Th1 immune response. The subject of the invention is also a method for raising an immune response in an individual, in particular in a human, wherein the obtained Th1 immune response is enhanced, comprising the step of administering to the individual in need thereof an immunologically effective amount of a composition of the invention.

Preparation of vaccine compositions

Advantageously, the polyacrylic acid polymer or polyacrylic acid polymer salt is purified, e.g. diafiltered, prior to addition to the vaccine composition. More specifically, it is purified, e.g., diafiltered, and then sterilized prior to its introduction into the vaccine composition. The sterilization can be performed by sterilizing the filtration or preferably by autoclaving.

According to the invention, the vaccine composition may be prepared by simply mixing a polyacrylic acid polymer salt, particularly in liquid form in aqueous solution or in aqueous buffered solution, with a suspension of the vaccine agent and other ingredients that may be present in the composition. This can be done as follows: the one or more selected vaccine agents are added on the polyacrylic acid polymer salt, in particular in the form of a liquid formulation in aqueous solution or in buffered aqueous solution, or the polyacrylic acid polymer, in particular in the form of a liquid formulation in aqueous solution or in buffered aqueous solution, is added on the suspension already containing the selected vaccine agent. On the other hand, in the case where it is desired to formulate a vaccine composition comprising a plurality of vaccine agents, it is preferred to first mix the polyacrylic acid polymer salt with one or more vaccine agents and thereafter incorporate the other ingredients.

Alternatively, if the vaccine agent is formulated as a freeze-dried or lyophilized product, the vaccine composition can be obtained by directly rehydrating the lyophilizate with a formulation (aqueous solution or buffered aqueous solution) containing a polyacrylic acid polymer salt.

The present invention also relates to the use of a polyacrylic acid polymer salt as defined herein, whether or not said embodiment relates to the "characteristics of polyacrylic acid polymer" in the above paragraph, for the preparation of a vaccine composition comprising at least one vaccine agent.

It is also an object of the present invention to carry out a method of mixing a vaccine composition of a polyacrylic acid polymer salt as defined in the present invention with at least one vaccine agent, whether or not said embodiment relates to the "characteristics of polyacrylic acid polymer" in the above paragraph.

For convenience, certain terms used in the specification, examples and appended claims are collected here.

As used herein, the term "animal" includes all vertebrates, including humans. It also includes individual animals at various stages of development, including embryonic and fetal stages. In particular, the term "vertebrate" includes, but is not limited to, humans, canines (e.g., dogs), felines (e.g., cats); as used herein, the term "avian" refers to any species or subspecies of the taxonomic group ava, such as, but not limited to, chickens (breeders, broilers and laying hens), turkeys, ducks, geese, quails, pheasants, parrots, sparrows, hawks, crows and ratites, including ostriches, pyrotechnics and ratites.

As used herein, the term "virulence" refers to an isolate that retains its ability to be infectious in an animal host.

As used herein, the term "inactivated vaccine" refers to a vaccine composition containing an infectious organism or pathogen that is no longer capable of replicating or growing. The pathogen may be of bacterial, viral, protozoan or fungal origin. Inactivation may be accomplished by a variety of methods, including freeze-thawing, chemical treatment (e.g., treatment with formalin), sonication, radiation, heat, or any other conventional means sufficient to prevent the organism from replicating or growing while retaining its immunogenicity.

The term "immunogenic" as used herein refers to the ability to generate an immune response against an antigen in a host animal. This immune response forms the basis of protective immunity elicited by vaccines against specific infectious organisms.

As used herein, the term "immune response" refers to a response elicited in an animal. The immune response may refer to cellular immunity (CMI); humoral immunity or possibly both. The invention also contemplates responses that are limited to a portion of the immune system. For example, the vaccine composition of the present invention can specifically induce an increased gamma interferon response.

As used herein, the term "antigen" or "immunogen" refers to a substance that induces a specific immune response in a host animal. Antigens may include whole organisms, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert having immunogenic properties; a DNA fragment or fragment capable of inducing an immune response when presented to a host animal; proteins, polypeptides, peptides, epitopes, haptens, toxins, antitoxins; or any combination thereof.

As used herein, the term "multivalent" refers to a vaccine comprising more than one antigen, whether from the same species (i.e., different isolates of the FMD virus serotype), from different species (i.e., isolates from Pasteurella haemolytica (Pasteurella haemeytica) and Pasteurella multocida), or a combination of antigens from different genera (e.g., a vaccine comprising a combination of antigens from Pasteurella multocida, salmonella (Salmonella), escherichia coli (Escherichia coli), haemophilus somnus), and Clostridium (Clostridium).

As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable vehicle" are interchangeable and refer to a fluid vehicle containing vaccine antigens that can be injected into a host without side effects. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions. The carrier may include adjuvants including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffers, viscosity enhancing additives, colorants, and the like.

As used herein, the term "vaccine composition" includes at least one antigen or immunogen in a pharmaceutically acceptable vehicle, which can be used to induce an immune response in a host. The vaccine composition may be administered by dosages and techniques well known to those skilled in the medical or veterinary arts, taking into account factors such as the age, sex, weight, species and condition of the recipient animal and the route of administration. The route of administration may be transdermal, by mucosal administration (e.g. oral, nasal, anal, vaginal) or by parenteral route (intradermal, intramuscular, subcutaneous, intravenous or intraperitoneal). The vaccine compositions may be administered alone, or may be co-administered or sequentially administered with other treatments or therapies. Forms of administration may include suspensions, syrups or elixirs, as well as preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g. by injection), for example sterile suspensions or emulsions. The vaccine composition may be administered as a spray or mixed in food and/or water or delivered in admixture with a suitable carrier, diluent or excipient (e.g., sterile water, physiological saline, dextrose, etc.). The compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents and the like, depending on the route of administration and the desired formulation. Suitable formulations can be prepared without undue experimentation by reference to standard Pharmaceutical texts, such as "Remington's Pharmaceutical Sciences", 1990.

Immunogens or antigens suitable for use in the present invention may be selected from inactivated pathogens, attenuated pathogens, immunogenic subunits (e.g., proteins, polypeptides, peptides, epitopes, haptens) or recombinant expression vectors, including plasmids with immunogenic inserts. In one embodiment of the invention, the immunogen is an inactivated or killed microorganism. In another embodiment of the invention, the vaccine composition comprises an immunogen selected from avian pathogens, including but not limited to Salmonella typhimurium (Salmonella typhimurium), salmonella enteritidis (Salmonella enteritidis), infectious Bronchitis Virus (IBV), newcastle Disease Virus (NDV), egg drop syndrome virus (EDS) or Infectious Bursal Disease Virus (IBDV), avian influenza virus and combinations thereof.

Alternatively, the vaccine composition comprises an immunogen selected from feline pathogens, such as Feline Herpes Virus (FHV), feline Calicivirus (FCV), feline leukemia virus (FeLV), feline Immunodeficiency Virus (FIV), rabies virus and combinations thereof.

In another embodiment, the vaccine composition of the present invention comprises an immunogen selected from canine pathogens including, but not limited to, rabies virus, canine Herpes Virus (CHV), canine Parvovirus (CPV), canine coronavirus, leptospira canicola (Leptospira canicola), leptospirovirus (Leptospira icohaemorragiae), leptospira grippotypa (leptospora grippotypa), bordetella bronchiseptica (Borrelia burgdorferi), bordetella bronchiseptica (Bordetella broncheseptica), and the like, and combinations thereof.

In another embodiment of the invention, the composition comprises an immunogen selected from equine pathogens, such as equine herpes virus (type 1 or type 4), equine influenza virus, tetanus, west nile virus, and the like, or combinations thereof.

In another embodiment of the invention, the composition comprises an immunogen selected from bovine pathogens, such as Foot and Mouth Disease Virus (FMDV), rabies virus, bovine rotavirus, bovine parainfluenza virus type 3 (bPIV-3), bovine coronavirus, bovine Viral Diarrhea Virus (BVDV), bovine Respiratory Syncytial Virus (BRSV), infectious bovine rhinotracheitis virus (IBR), escherichia coli, pasteurella multocida (p.multocida), pasteurella haemolytica (p.haemolytica), and combinations thereof.

In another embodiment of the invention, the composition comprises a vaccine agent comprising an immunogen selected from porcine pathogens such as, but not limited to, swine Influenza Virus (SIV), porcine circovirus type 2 (PCV-2), porcine reproductive respiratory syndrome virus (PRRS), pseudorabies virus (PRV), porcine Parvovirus (PPV), FMDV, mycoplasma hyopneumoniae (m. Hyopnemoniae), erysipelothrix rhusiopathiae, pasteurella multocida (Bordetella bronchiseptica), escherichia coli, and the like, and nucleic acids encoding the immunogen, and combinations thereof.

Vaccine agents comprising viruses, bacteria, fungi, etc. may be produced by in vitro culture methods using appropriate media or host cell lines and conventional methods well known to those of ordinary skill in the art. For example, PRRS can be cultured in a suitable cell line, such as the MA-104 cell line (see U.S. Pat. nos. 5,587,164. In a similar manner, PCV-2 can be cultured using a PK-15 cell line (see U.S. Pat. No.6,391,314); SIV can be cultured on eggs (US 6,048,537); and Mycoplasma hyopneumoniae can be cultured in a suitable medium (U.S. Pat. No. 5,968,525; U.S. Pat. No. 5,338,543).

To obtain an inactivated immune or vaccine composition, the pathogen is preferably inactivated after harvesting and optionally clarified by chemical treatment with e.g. formalin or formaldehyde, beta-propiolactone, ethylenimine, binary Ethylenimine (BEI), and/or physical treatment (e.g. heat treatment or sonication). Inactivation methods are well known to those skilled in the art. For example, FMD virus can be inactivated by ethyleneimine (Cunliffe, HR, applied Microbiology,1973, p.747-750) or by high pressure (Ishimaru et al, vaccine 22 (2004) 2334-2339) and PRRS virus can be inactivated by beta-propiolactone treatment (R) ((R) ())Plana-Duran et alVet. Microbiol, 1997, 55: 361-370) or by BEI treatment (US 5,587,164); PCV-2 virus can be inactivated using ethyleneimine treatment or by β -propiolactone treatment (U.S. Pat. No.6,391,314); swine influenza virus can be inactivated using detergents such as Triton or by treatment with formaldehyde (US 6,048,537); hyponeumoniae can be treated with formaldehyde (RossR.F.Above), ethyleneimine or BEI treatment.

The inactivated pathogen may be concentrated by conventional concentration techniques, particularly by ultrafiltration, and/or purified by conventional purification methods, particularly using chromatographic techniques, including but not limited to gel filtration, ultracentrifugation using a sucrose gradient, or selective precipitation, particularly in the presence of PEG.

Immunogens that may be used in the vaccine compositions of the present invention also include expression vectors. Such vectors include, but are not limited to, in vivo recombinant expression vectors, such as polynucleotide vectors or plasmids (EP-A2-1001025;Chaudhuri Pres.vet.sci.2001, 70: 255-6), viral vectors such as, but not limited to, adenoviral vectors, poxvirus vectors such as fowlpox (US 5,174,993; US5,505,941; and US5,766,599) or canarypox vector (US 5,756,103) or bacterial vector (escherichia coli or salmonella).

The invention also includes formulations of multivalent immunization compositions or combination vaccine compositions. For example, antigens that may be used in combination with bovine bacteria prepared according to the present invention include, but are not limited to, mycoplasma bovis (Mycoplasma bovis), pasteurella (Pasteurella sp.), in particular Pasteurella multocida and Pasteurella haemolytica, haemophilus, in particular haemophilus somni, clostridium, salmonella, corynebacterium (Corynebacterium), streptococcus, staphylococcus (Staphylococcus), moraxella (Moraxella), escherichia coli, and the like.

The invention further provides a method of inducing an immune response in a host (e.g., an animal) comprising administering to the host an immunological composition or a vaccine composition of the invention. The immune response elicited in this way is in particular an antibody and/or cellular immune response, and in particular a gamma interferon response.

In particular, the invention provides methods of immunizing against or preventing or alleviating the symptoms caused by infection of an animal by a pathogenic organism (e.g., infection by a virus, bacterium, fungus, or protozoan parasite). The methods of the invention are useful in vertebrates, including but not limited to humans, canines (e.g., dogs), felines (e.g., cats); equine (e.g., horse), bovine (e.g., cow), and porcine (e.g., pig); and poultry, including but not limited to chickens, turkeys, ducks, geese, quail, pheasants, parrots, sparrows, hawks, crows, and ratites (ostriches, turkeys, crane ostriches, etc.).

In a particular aspect of the invention, the methods consist of vaccinating a pregnant female prior to delivery by administering a vaccine composition prepared according to the invention. These methods also include the induction of protective antibodies elicited by the vaccination protocol and the transfer of these protective antibodies from the vaccinated pregnant female to its offspring to protect the offspring from infection and disease.

The dosage of the vaccine composition prepared according to the present invention will depend on the species, breed, age, size, vaccination history and health status of the animal to be vaccinated. Other factors such as antigen concentration, other vaccine components and route of administration (i.e., subcutaneous, intradermal, oral, intramuscular or intravenous administration) will also affect the effective dose. The dose of vaccine administered can be readily determined based on the antigen concentration of the vaccine, the route of administration, and the age and condition of the animal to be vaccinated. Each batch of antigen can be calibrated individually. Alternatively, systemic immunogenicity assays at varying doses, as well as LD, may be used ₅₀ Studies and other screening procedures are conducted to determine effective dosages of the vaccine compositions of the present invention without undue experimentation. It is apparent from the examples given below what are the appropriate approximate doses and approximate volumes for using the vaccine compositions described herein. The key factor is that the dose provides at least partial protection against the natural infection, as evidenced by a reduction in mortality and morbidity associated with the natural infection. The appropriate volume can likewise be readily determined by one of ordinary skill in the art. For example, in avian species, the volume of the dose may be from about 0.1ml to about 0.5ml, and advantageously, from about 0.3ml to about 0.5ml. For feline, canine and equine species, the volume of the dose may be from about 0.2ml to about 3.0ml, advantageously from about 0.3ml to about 2.0ml, more advantageously, from about 0.5ml to about 1.0ml. For bovine and porcine species, the dosage may be from about 0.2ml to about 5.0ml, advantageously from about 0.3ml to about 3.0ml, more advantageously from 0.5ml to about 2.0ml.

It may be preferred to repeat the vaccination at periodic intervals to initially enhance the immune response, or when a significant period of time has elapsed since the last administration. In one embodiment of the invention, the vaccine composition is administered by parenteral injection (i.e. subcutaneously, intradermally or intramuscularly). The composition may be administered as one dose, or in the alternative, in repeated doses of about 2 to about 5 doses at intervals of about two to about six weeks, preferably at intervals of about two to five weeks. However, one skilled in the art will recognize that the number of doses and the time interval between vaccinations depends on a number of factors, including, but not limited to, the age of the vaccinated animal; a condition of the animal; (ii) an immunization route; the amount of antigen available per dose, etc. For initial vaccination, this period is typically longer than one week, preferably will be between about two and five weeks. For previously vaccinated animals, booster immunizations may be given before or during pregnancy at approximately one year intervals.

The invention further relates to a method of treating a host, such as an animal, comprising administering to the host a pharmaceutical composition prepared according to the invention comprising at least one immunogen selected from a protein or peptide, an inactivated or attenuated virus, an antibody, an allergen, a CpG ODN, a growth factor, a cytokine or an antibiotic, and in particular a CpG ODN or a cytokine. These pharmaceutical compositions are useful for improving the growth performance of an animal (e.g., a chicken, pig, cow, or cow).

In one embodiment, the present disclosure provides an immunological or vaccine composition comprising an adjuvant formulation, a therapeutically effective amount of an antigenic component and a pharmaceutically or veterinarily acceptable carrier, wherein the adjuvant formulation comprises a non-crosslinked polyacrylic acid (PAA) polymer having a weight average molecular weight (AMw) of about 350kDa to about 650 kDa. In some embodiments, the antigenic component can comprise an attenuated recombinant viral vector, a native or genetically engineered attenuated live virus or microorganism, an inactivated virus or microorganism, a coccidia precocious microorganism, a protein subunit, a unicellular parasite, a multicellular parasite, or any combination of the foregoing.

In one embodiment, the antigenic component may comprise: canine Coronavirus (CCV) antigen, canine Distemper Virus (CDV) antigen, canine parvovirus antigen (CPV), canine parainfluenza virus (CPI) antigen, feline Calicivirus (FCV) antigen, feline Immunodeficiency Virus (FIV) antigen, feline Herpes Virus (FHV) antigen, feline leukemia virus (FeLV) antigen, cancer antigens (e.g., her2-neu, tyrosinase, IL-2, etc.), eimeria sp or antigen thereof, escherichia coli or antigen thereof, mycoplasma hyopneumoniae (m.hyo), bovine Diarrhea Virus (BDV) antigen, a surface protective antigen (SpaA) from Erysipelothrix rhusiopathiae, a recombinant canarypox vector containing and capable of expressing at least one protective immunogen in vivo, inactivated full-length rabies glycoprotein, erysipelothrix sp, erysipelothrix rhusiopathiae, a surface protective antigen (SpaA) from Erysipelothrix rhusiopathia), a fusion protein comprising at least a portion of at least one additional immunogen, a fusion protein of SpaA, a fusion protein, clostridium perfringens toxin (clostridium perfringens), clostridium perfringens C toxin (clostridium perfringens).

In another embodiment, the antigenic component comprises or consists of inactivated full length rabies glycoprotein. The antigenic component can also comprise a clostridium perfringens B/C toxin, a clostridium perfringens D toxin, a clostridium septicum toxin, a clostridium nokohlii toxin, a clostridium tetani toxin, or a combination thereof. In a particular embodiment, the immunization or vaccine composition may comprise an antigenic component comprising a clostridium perfringens B/C toxin, a clostridium perfringens D toxin, a clostridium septicum toxin, a clostridium noffii toxin, and a clostridium tetani toxin. The PAA adjuvants disclosed herein provide "dose retention".

In another embodiment, the antigenic component comprises a SpaA antigen or a fusion protein comprising a SpaA antigen.

In another embodiment, the antigenic component comprises an attenuated fowlpox virus or a DNA plasmid containing and capable of expressing an influenza virus gene in vivo. The antigenic component may also comprise an attenuated fowlpox virus or a DNA plasmid containing and capable of expressing the rabies glycoprotein gene in vivo.

In another aspect, various methods of treatment are provided. For example, the present disclosure provides a method of treating a bovine against an infection caused by a bacterium, the method comprising administering to the bovine a vaccine composition comprising a PAA polymer having a Mw in the range of about 350kD to about 650 kDa. In particular embodiments, PAA having a Mw of about 450kDa is particularly useful for eliciting protective immune responses in animals, including bovids.

In one embodiment, the present disclosure provides a method of treating a canine or equine animal against infection by influenza, the method comprising administering to the canine or equine a vaccine comprising fowlpox virus or a DNA plasmid containing and capable of expressing influenza antigens in vivo. In a specific embodiment, the influenza antigen is an HA gene.

In another embodiment, the present disclosure provides a method of treating a canine against infection by rabies virus, the method comprising administering to the canine a vaccine composition comprising inactivated rabies glycoprotein and PAA having a Mw of about 350kDa to 650 kDa.

The present invention also provides avian vaccines, including avian coccidiosis vaccines, for in ovo administration, which may comprise:

an adjuvant comprising non-crosslinked PAA having an average Mw of about 350kDa to about 650 kDa; and

a protozoan antigen selected from the group consisting of (1) one or more recombinantly expressed proteins; (2) One or more proteins or other macromolecules isolated from the protozoa by conventional methods; (3) A whole cell extract or preparation from said protozoa; and (4) inactivated, live or live precocious coccidia selected from the group consisting of: eimeria acervulina (Eimeria (e), eimeria acervulina), eimeria adenoids (e.adeneides), eimeria brunetti (e.brunetti), eimeria colchica (e.colchici), eimeria curvata (e.currvata), eimeria dispersa (e.dispersa), eimeria dodecaalis (e.duodenalis), eimeria castellata (e.fratercula), eimeria gabonensis (e.galopovaes), eimeria innocua (e.incocua), eimeria praecox (e.praecox), eimeria maxima (e.maxima), eimeria perviviparii (e.melela), eimeria perreas and Eimeria mitis (e.mitis), eimeria perlecanii (e.mitis), eimeria acervulina (e.mitis), eimeria acervulineria acervulina (e.mitis), eimeria acervulina (e.mitis).

In a specific embodiment, the PAA has an average Mw of about 450 kDa.

The invention further provides a method of treating a bovine against infection by escherichia coli or mycoplasma hyopneumoniae, the method comprising administering to the bovine a vaccine composition comprising PAA and escherichia coli or mycoplasma hyopneumoniae. The invention necessarily comprises a method of treating a pig against an infection caused by mycoplasma hyopneumoniae, the method comprising feeding the pig with a vaccine comprising mycoplasma hyopneumoniae.

In another embodiment, the immunological or vaccine composition comprises an antigen corresponding to an agent responsible for infection and/or disease state in a feline. The antigen may comprise Feline Immunodeficiency Virus (FIV). The invention also provides a method of treating an infection caused by FIV in a feline comprising administering to the feline a vaccine comprising FIV antigen and PAA.

In another embodiment, the present disclosure provides a vaccine composition comprising a cancer antigen. In related embodiments, the present disclosure provides a method of treating a subject against cancer, the method comprising administering to the subject a vaccine composition comprising a cancer antigen and PAA.

The properties and advantages of the polymers used in the present invention are highlighted in the examples below with reference to the drawings. In these examples, naPAA represents the sodium salt of the polymer, whether or not it is according to the invention.

When not specified, "molecular weight" means "weight average molecular weight".

Example 1 materials and methods for characterizing polymers

All determinations of Mw, mark-houwink slope, IP, monomer and persulfate contents were made according to the methods below.

I.1-Mw, mark-Houwink slope and IP

1. Chemicals and reagents

The water was purified using a Milli-Q-UF system (Millipore, milford, MA, USA). Phosphate buffered saline (PBS 1c ^-1 Na ₂ HPO ₄ ，2H ₂ O；1.5mMol.L ^-1 KH ₂ PO ₄ ；2.7mMol.L ^-1 KCl；137mMol.L ^- ¹ NaCl; ph 6.8). The assay was carried out with DNA from Sigma Aldrich (Saint Quantin Fallavier, france) (CAS number 73049-39-5) and sucrose from VWR (Darmstadt, germany) (CAS number 57-50-1)And determining the gel permeation characteristic. Standards used for system calibration were Pullulan 100KDa from Malvern (Malvern, UK) and Pullulan 400KDa from Agilent (Santa Clara, calif.).

2. Size Exclusion Chromatography (SEC) with triple detection

A Viscotek GPCmax VE2501 system (Malvern Instrument, malvern, UK) containing an HPLC pump with a built-in degasser and an autosampler with a 100 μ L injection ring was used for HP-SEC analysis. The Viscotek TDA302 detector system has a refractive index, right angle light scattering and four-capillary differential viscometer detector for on-line SEC signal detection. The order of the detectors is as follows: LS (right angle light scattering) -RI (refractive index) -VIS (four-capillary differential viscometer). A 0.22 μm nylon pre-filter was placed between the column and the detector. The OmniSEC 4.7 software program was used to acquire and analyze SEC data. All detectors were calibrated in the mobile phase in PBS1C with 100KDa amylopectin standard. The PBS1C mobile phase was filtered through a 0.22 μm microporous nitrocellulose filter and degassed prior to use. Separation of the NaPAA samples was achieved by two A6000M (8 mm ID. Times.30 cm L) columns (Malvern) connected in series. At 0.6mL.min ^-1 The elution is isocratic at the flow rate of (2). Measured at about 0.4mg.ml for Mw, IP and mark-houwink slope ^-1 Target NaPAA concentration of (2) was injected into 100. Mu.L of sample. The pullulan 400kDa standard was used as a "control" sample for all analyses. Void volume (V) of the column ₀ ) And total permeate volume (V) _t ) Determined by injecting high molecular weight DNA and sucrose, respectively.

3. Preparation of standards and samples

By dissolving the starting material in PBS1C to final concentrations of1, 0.1 and 2mg.mL, respectively ^-1 To prepare pullulan, DNA and sucrose standards. NaPAA was formulated and diluted at the target concentration in PBS 1C.

Determination of NaPAADn/dc values and Mw, mark-Houwink slope and IP measurements

The dn/dc coefficient is related to the molar mass according to the following relationship (Zimm, 1948, j. Chem. Phys.,16, 1099-1116):

where K is an optical constant comprising dn/dc of a particular scattering system described in the equation below, C is the concentration of NaPAA in the sample, R is _θ Excess intensity of light scattered at an angle θ, mw is the weight average molecular weight, and A ₂ Is the second coefficient of Virial coefficient, which can be considered to be zero since the concentration of each sample fraction is very low. P (θ) is a particle scattering function representing the angular dependence of the light scattering intensity, and is associated with the radius of gyration (Rg) of the polymer molecules.

Wherein n is ₀ Is the refractive index of the solvent in the sample; n is a radical of hydrogen _A Is the Avogastron constant; lambda [ alpha ] ₀ Is the wavelength of the laser beam in the vacuum.

For sizes smaller than λ ₀ For the small molecule of/20, it is estimated that the intensity of scattered light is independent of the scattering angle, and therefore P (θ) =1 for all angles.

The expression now obtained:

R _θ ＝KCM _W

dn/dc of NaPAA by OmnisSEC software, from 0.4 to 1mg.mL using a triple detection system with addition of NaPAA and SEC of known concentration ^-1 And (4) automatic calculation. Each concentration was injected in duplicate. The experiment was repeated under the same conditions with different representative NaPAA batches of known concentration. The final dn/dc coefficient corresponds to the average of these measurements and is determined to be 0.172mL ^-1 . The dn/dc value was used for further molecular weight determination.

The Mw, IP and Mark-Houwink slope determinations were made with solutions of polyacrylic acid polymer salts in PBS1C having known concentrations of polyacrylic acid polymer salts, e.g., 0.4mg.mL ^-1 . Since the polyacrylate salt represents more than 95% by dry weight of the polyacrylic acid polymer salt, the weight of dry matter is considered to be polyacrylic acidDry weight of compound salt.

The Intrinsic Viscosity (IV) of NaPAA is related to its molecular weight and conformation; and is represented by a mark-houwink diagram:

IV＝K·MW ^a

where "a" and "K" are constants that specify the solute-solvent system, and the "a" index varies between 0 (solid spheres) and 2 (rods).

Mark-Houwink slope "a" according to the formula IV = K.MW ^a And (4) determining.

A slope "a" greater than or equal to 0.7 means that the NaPAA can be considered linear.

Defining the polydispersity Index (IP) as Mw/M _n And Mn is the number average molecular weight. The Mn of NaPAA was automatically calculated by OmniSEC software.

I.2 determination of persulfate and acrylate monomers

A. In the raw materials

1. Chemicals and reagents

The water was purified using a Milli-Q-UF system (Millipore, milford, mass.). 25mM (phase A) and 200mM (phase B) sodium hydroxide solutions were prepared using 46-51% concentrated sodium hydroxide from Fisher Scientific (Illkirch, france). 0.1N sodium hydroxide is from VWR (Darmstadt, germany). The standards used for calibration were sodium persulfate from Fisher Scientific (ilkirch, france) and sodium acrylate from Sigma Aldrich (Saint quantin Fallavier, france). The internal standard used to correct for sample preparation variation was sodium oxalate from Sigma Aldrich (Saint quantin Fallavier, france).

2. High performance anion exchange chromatography using conductivity detection

Acrylate and persulfate impurities in a NaPAA sample were simultaneously detected by High Performance Anion Exchange Chromatography (HPAEC) using conductivity detection. An ICS-3000 (Dionex, thermo Fisher Scientific, pittsburgh, pa.) ion chromatography system was used. It was equipped with an SP-1 pump, a thermostatted autosampler (5 ℃), a thermostatted column (40 ℃) and a conductivity detector compartment (30 ℃). An ATC3RFIC (9 x24 mm) carbonate trap column (Thermo FisherScientific) was placed in front of the column to trap the carbonate anions and improve the overall analytical sensitivity. Analytical separation was achieved on an anion exchange AS-11HC column (250X 4 mM) from Dionex (Thermo Fisher Scientific) with a gradient elution of 25mM (phase A) to 200mM (phase B) sodium hydroxide solution. The gradient program was: 0% B (12 min), 0-40% B (5 min), 40-100% B (8 min), 100% B (25 min), 100-0% B (1 min), 0% B (9 min). The flow rate of the mobile phase was 1mL/min and the injection volume was 50. Mu.L. An AG-11HC pre-column (50X 4 mm) from Dionex (Thermo Fisher Scientific) was used to protect the analytical column. A Dionex conductivity suppressor (AERS 082540, thermo Fisher Scientific) was placed in front of the detector compartment to improve the signal. Under these chromatographic conditions, the retention times for the acrylate, oxalate and persulfate salts were 4, 11 and 45 minutes, respectively.

3. Preparation of samples

Prior to chromatography, high molecular weight species were removed from the sample. Briefly, a NaPAA sample was diluted 10-fold with water (resulting in a concentration of 10mg/mL in NaPAA) and an internal sodium oxalate standard was added to achieve a concentration of 50. Mu.g/mL. Then 500. Mu.l of the sample was centrifuged at 14000g for 30 minutes, sequentially through Amicon Ultracel 0.5ml-100k and Amicon Ultracel 0.5ml-3k centrifugal filters from Merck Millipore (Darmstadt, germany). Before use, the Amicon centrifugal filter was washed sequentially with water and 0.1N NaOH to remove glycerol.

4. Calibration and results

Quantification of persulfate and acrylate impurities was performed by external calibration prepared with commercial standards. Six concentrations of 1-100. Mu.g/mL sodium acrylate and sodium persulfate were mixed in water and 200. Mu.L of 500. Mu.g/mL sodium oxalate internal standard was added to each concentration. The calibration curve follows a linear model for persulfate and a quadratic model for acrylate. The impurity content is expressed in% w/w (wt% of acrylate or persulfate impurity relative to dry NaPAA weight) or μ g of persulfate or acrylate impurity per gram of starting NaPAA.

B. In the NaPAA polymer obtained after purification

Persulfate and residual acrylic acid impurities in the NaPAA sample after the purification step (by dialysis, ultrafiltration, or gel filtration) were determined as described above for the assay in the NaPAA starting sample. However, in the purified samples, the 10-fold dilution step was omitted from the sample preparation procedure. For persulfate, the chromatographic conditions were the same as for the starting material, naPAA, and the analysis was managed as a limit test with a detection limit of 100ng/mL. For the acrylates, the same HPAEC system was used, but analytical separation was achieved on an anion exchange carbopac SA10 column (250 x4 mM) from Dionex (Thermo Fisher Scientific) eluting with a gradient of 30mM (phase a) to 200mM (phase B) sodium hydroxide solution. The gradient program was: 0% B (14 min), 0-100% B (6 min), 100% B (15 min), 100-0% B (1 min), 0% B (9 min). The flow rate of the mobile phase was 1mL/min and the injection volume was 50. Mu.L. A CarboPacTM SA10G column (50X 4 mm) from Dionex (Thermo Fisher Scientific) was used to protect the analytical column. A Dionex conductivity inhibitor (AERS 082540, thermoFisher Scientific) was placed in front of the detector chamber to improve the signal. Under these chromatographic conditions, the retention time of the acrylate was about 11 minutes. Residual acrylic acid impurities were determined from a linear calibration curve constructed in PBS1C using external acrylic acid standards at 20-500 ng/ml. The results are expressed above as% w/w (wt% acrylate or persulfate impurity relative to dry NaPAA weight%) or μ g of persulfate or acrylate impurity per mL of NaPAA adjuvant solution.

Example 2 testing of adjuvant Activity

1) Adjuvant for the association of PAA of the present invention with staphylococcus aureus antigen compared to 2 PAA of the prior art Evaluation of Effect

The adjuvant activity OF the polymer-based formulation was tested in distantly hybridizing OF1 mice using type 5 polysaccharide from staphylococcus aureus (PS 5) conjugated to recombinant exotoxin a OF Pseudomonas aeruginosa (rEPA) as a model antigen.

The antigen was prepared as follows:

staphylococcus aureus (Reynolds strain) was grown in SATA-1 broth with stirring (100 rpm) for 72 hours and then inactivated by the addition of 1/1 (v/v) phenol/ethanol solution to the endConcentration was 2% w/v. Bacterial cells were pelleted at 16000g for 75 minutes. The cell paste was suspended at 0.5g (wet weight) in 50mM Tris-2mM MgSO ₄ pH 7.6/ml. Lysostaphin (100. Mu.g/ml) was added and the suspension was incubated at 37 ℃ for 4 hours with stirring. Subsequently, benzonase was added at a concentration of 5U/ml and incubation was continued for 2 hours. The reaction mixture was concentrated by tangential flow filtration (molecular weight cut-off 30000 Da). The resulting concentrate was digested with benzonase (5U/ml) for 6 hours at 37 deg.C and then with pronase (4U/ml) in the appropriate buffer (Tris 50mM, pH 8.0, containing 1mM MgCl ₂ And 1mM CaCl ₂ ) Digestion was carried out at 37 ℃ for 15 hours. After centrifugation at 5000g for 30 min, the supernatant was concentrated by tangential flow filtration (30,000da MWCO). The solution was then adjusted to a final concentration of 50mm tris hcl, ph 7.5. Aliquots were loaded onto a Q Sepharose (20 mL) column equilibrated with 50mM Tris HCl, pH 7.5. The column was eluted at a flow rate of 2ml/min and fractions of 5ml were collected. The column was washed with 5 volumes of starting buffer. The polysaccharide was then eluted with a linear gradient of 250ml of 0 to 0.5M NaCl in 50mM Tris HCl, pH 7.5. The fractions containing polysaccharide and teichoic acid were dialyzed and freeze-dried as detected by 210nm optical absorption and high performance anion exchange chromatography (HPAEC-PAD).

The purified polysaccharide was then activated in NaCl by Adipic Dihydrazide (ADH). The pH was adjusted to 4.9 and Ethyl Dimethyl Aminopropyl Carbodiimide (EDAC) was added. As activation proceeded for 90 minutes at ambient temperature, the pH was constantly adjusted to 4.9 with 0.1N HCl. The reaction was terminated by the addition of NaOH until neutralization (pH 7.0). The activated polysaccharide was then dialyzed against 500mM NaCl in water and then against water. The activated and dialyzed polysaccharide was then lyophilized. The percent functionalization was estimated to be about 5.9% (w/w).

The activated polysaccharide solution was mixed with carrier protein (rEPA) in NaCl and EDAC. Conjugation was performed at 4 ℃ and the pH was maintained at 5.7 by addition of 0.1N HCl. After 90 minutes, the reaction was stopped by adding 0.2N NaOH until pH7. The conjugated antigen was then dialyzed against aqueous NaCl and then purified by size exclusion chromatography on a 200mM equilibrated agarose gel Cl-4B column. NaCl in 10mM phosphate buffer, pH 7.2. The fractions containing the conjugate (detected by optical absorption at 206nm and 278 nm) were pooled and eluted mainly with the dead volume of the column.

The polymer formulation was prepared as follows:

a product named PAA225000 (ref.18613, sodium salt) was obtained from Polysciences Europe (Eppelheim, germany) in the form of a concentrated solution. It was diluted with water to a concentration of 20mg/ml and stirred at room temperature for 12 hours. The pH was adjusted to 7.55 with HCl and the solution was dialyzed against 150mM NaCl aqueous solution (3 successive baths) at room temperature by using a 2kDa cut-off dialysis cartridge (Thermo Fischer Scientific, courtaloeuf, france). The solution was then filtered through a 0.22 μm pvdf membrane for sterilization. The molecular weight of the polymer salt was then measured to be 488 550Da. Its Mn is 129 070Da and its IP is 3.8.

The polymer was then stored at +4 ℃ as a solution containing 20mg/ml polymer in 150mM NaCl in water. This solution was then mixed with PBS1C and concentrated 10 times with sterile water to give a saline solution containing 2mg/ml of the polymer salt.

The product named PAA20 is obtained as sodium salt in dry powder form from Polymer Expert (Pessac, france). It was rehydrated in water to a concentration of 20mg/ml and stirred at room temperature for 12 hours. The solution was then filtered through a 0.22 μm PVDF membrane for sterilization. The molecular weight of the polymer salt was measured at 100 700Da. The Mn was 46 700Da and the IP was 2.2. The polymer was then stored at +4 ℃ as a solution containing 20mg/ml of polymer salt in 150mM NaCl in water. This solution was then mixed with PBS 10C and sterile water to give an aqueous salt solution containing 2mg/ml of the polymer salt.

974P (called ^ er)>

) Which is a reticulated PAA polymer, with a molecular weight of several million Da, is diluted with PBS to give a solution containing 2mg/ml of polymer.

An adjuvant formulation to be injected into an animal is prepared by mixing the antigen solution and the polymer solution vol/vol. Each injection dose had 200. Mu.g of polymer and 2.5. Mu.g of polysaccharide in PBS1C solution.

And (3) immunization:

with PAA20, PAA225000 or

Groups OF 5 to 10 OF1 mice aged 7 to 9 weeks were immunized alone (these served as negative controls) or with a formulation comprising antigen and adjuvant. One group of mice was injected with PS5-rEPA alone. The dose was administered by the Subcutaneous (SC) route in the scapular region of D0, D21 and D35. Blood samples were collected on D42 for immune response analysis.

Blood samples were collected in vacuum tubes containing clotting activator and serum separation gel (Becton Dickinson, meylan, france). Tubes were centrifuged at 2600g for 20 min to separate serum from cells. Sera were transferred to deep well plates and heat inactivated at 56 ℃ for 30 min, then stored at-20 ℃ until they were used for subsequent assays.

The test includes the following different groups:

PBS

PAA20

PAA225000

PS5-rEPA

PS5-rEPA+PAA20

PS5-rEPA+PAA225000

blood samples were used to test specific IgG1 and IgG2a antibodies produced by the immunized mice.

The ELISA test used the activated polysaccharide PS5 (PS 5 conjugated to ADH: PS 5-AH) as the coated antigen. Briefly, ELISA plates were coated with 100. Mu.L per well of activated polysaccharide solution in 1. Mu.g/mL PBS 1C. The plates were incubated at 4 ℃ for 12 hours and emptied by inverting the plates. The wells were blocked by adding 150. Mu.L of saturation buffer 1 (PBS 1C/Tween 0.05% w/v/bovine albumin 1% w/v) and incubated for 1 hour at 37 ℃. The ELISA plate was emptied by inversion. Two-fold serial dilutions (12 times) of each serum were performed directly in ELISA plates using buffer 1 as the dilution buffer, with a final volume of 100 μ Ι _ per well. Plates were incubated at 37 ℃ for 90 minutes and then washed 3 times (250. Mu.L per well) with buffer 2 (PBS 1C/Tween 0.05% w/v). Anti-mouse IgG1 or anti-mouse IgG2a peroxidase conjugate was diluted in buffer 1 (1/8000) and used as a secondary antibody (100. Mu.L per well). After incubation at 37 ℃ for 90 minutes, the ELISA plates were washed 3 times with buffer 2 (250. Mu.L per well). The reaction was developed by adding 100. Mu.L of tetramethylbenzidine substrate solution to each well, and stopped after 30 minutes at room temperature with 100. Mu.L/well of 1N Cl. The absorbance was measured at 450-650 nm. Antibody titers were expressed in arbitrary units corresponding to reciprocal serum dilutions of OD450nm =1 using SoftmaxPro software.

The results obtained are summarized in fig. 1 and shown:

1) No background was measured in negative control sera (< 1.3 Log) from mice immunized with PBS1C or polymer-based formulation alone (data not shown).

2) After a third immunization with PS5-rEPA conjugate only, a specific immune response against PS5 polysaccharide was obtained, mainly against PS5IgG1 (4.8 Log). The anti-PS 5 antibody response elicited by PS5-rEPA conjugate injected alone was predominantly Th2 driven, with IgG1/IgG2a ratios approaching 126.

3) When PS5-rEPA conjugates with

PAA20 or PAA225000 were formulated together with no increase in anti-PS 5IgG1 titers, with an average anti-PS 5 titer of approximately 4.8Log. />

Or co-injection of PAA20 resulted in increased anti-PS 5IgG2a titers, but these anti-PS 5 antibody responses were still predominantly Th2 driven,the IgG1/IgG2a ratios were close to 50 and 63, respectively. A strong increase in anti-PS 5IgG2a titer (4.5 Log) was observed when the PS5-rEPA conjugate was co-injected with PAA225000. Co-injection of PAA225000 elicited a Th1 biased anti-PS 5 antibody response with an IgG1/IgG2a ratio close to 2.

In summary, this experiment demonstrates that while the antigen alone induces predominantly a Th2 response, the adjuvants of the invention are able to induce a more pronounced Th-1 immune response without affecting the Th-2 response than the other polymers tested.

Blood samples were also tested for their ability to induce opsonic activity in human peripheral mononuclear cells (hPMN). For this test, pooled sera from each group were tested at serial 10-fold dilutions using a Lowenstein strain (ATCC 49521) cultured at 37 ℃ for 20 hours in TSB medium alone or supplemented (stationary phase).

Human peripheral blood from healthy human volunteers was collected in heparin sodium evacuated blood collection tubes. 10 ml is required for each donor. Leukocytes were isolated by lysing erythrocytes in ammonium chloride lysis buffer. The cells were washed twice in PBS1C and finally suspended in 5mL OPA medium (RPMI-Hepes supplemented with 0.5% BSA and 2mM glutamine). Large leukocytes (predominantly hPMN (95%)) were then counted on a Multisizer Coulter counter and conditioned to 0.25X 10 in OPA medium ⁶ Concentration in/mL.

After 20h growth, the bacteria were washed 2 times with PBS1C and resuspended at 5mLPBS 1C. The bacterial concentration was adjusted to 10 in OPA medium ⁸ CFUs/mL。

Oxidative burst assays were performed in 96-well polypropylene Deepwell plates. The plates were kept on ice after continuous addition of reagents. Reagents were added to the wells in the following order: mu.L of heat-inactivated specific serum, measured at dilutions ranging from 1/10 to 1/640, 250. Mu.L of bacteria, 50. Mu.L of baby rabbit complement, 1/10, 100. Mu.L of leukocytes and 50. Mu.L of LDHR (molecular probe, D632) at 1mg/mL. The final reaction volume was 500. Mu.L. The plates were then incubated at +37 ℃ with gentle shaking in the dark for 25 minutes. The final bacteria/large leukocyte ratio was 100, the final dilution of serum and complement was 1/100 to 1/6400, and the final concentration of dhr was 0.1mg/mL. At the end of the incubation period, the plate was placed on ice to stop the reaction. The analysis was performed on a Cytomics FC 500. The oxidized form of DHR, rhodamine 123, fluoresces brightly upon excitation at 488 nm. Gates were defined on large granular leukocyte populations on the (FSC/SSC) dot plots to differentiate PMN populations. Three thousand events were obtained from each hole on the door. Results are expressed as the percentage of fluorescence-activated PMNs (rhodamine 123 positive PMNs) in the entire PMN population.

The results are shown in table 1 below:

TABLE 1

¹ % activated PMNs/whole PMN population: + + + + (80% -100% at 1/1000 serum hygroscopicity)

+ + (80% -100% at 1/100 serum hygroscopicity); + (30% -70% under 1/100 serum hygroscopicity)

These results in table 1 show that:

1) No activated PMNs were detected with mice immunized with PBS or negative control sera injected with polymer-based formulation alone (data not shown).

2) The PS5-rEPA conjugate elicits anti-PS 5 serum antibodies, which weakly activate hPMN in the presence of bacteria. The percentage of activated hPMN that produces an oxidative burst is estimated to be 30% to 70% for 1/100 serum dilution.

3) Co-injection of PS5-rEPA with carbomer or PAA20 moderately increased the ability of anti-PS 5 serum antibodies to recognize the surface of the Staphylococcus aureus Lowenstein strain. The percentage of activated hPMN that produces an oxidative burst is estimated to be 80% to 100% for 1/100 serum dilutions.

4) Co-injection of PS5-rEPA with PAA225000 significantly improved (10-fold increase) the ability of anti-PS 5 serum antibodies to recognize the surface of the Staphylococcus aureus Lowenstein strain. The percentage of activated hPMN that produces an oxidative burst is estimated to be 80% to 100% for 1/1000 serum dilution.

They were also tested for their ability to kill staphylococcus aureus Lowenstein bacteria in the presence of human PMNs with sera obtained from PAA225000 and PAA20 or with sera obtained from mice immunized with antigen alone.

For this test, EFS (Etablessment) was passed

du Sang) whole blood was collected from sodium citrate bags of healthy human donors and fresh human polymorphonuclear leukocytes (PMNs) were isolated according to the following procedure. Erythrocytes were lysed by incubating 5mL blood and 45mL lysis buffer for 10 min at +20 ℃. The PMN was washed twice in HEPES saline buffer (HBSS, no CaMg, ref pH 7.4). PMNs survived more than 90% as shown by trypan blue exclusion. Dilution of PMN suspension to 10 ⁷ Individual cells/mL.

The Lowenstein Staphylococcus aureus strains were cultured in TSB medium for 12 hours. The bacterial cells were precipitated, washed with OPA medium (RPMI +5% SVF +0.05% Tween 20) and suspended to 5x10 in physiological saline ⁷ Individual cells/mL. The following were added to each tube: 0.25mL PMN, 50. Mu.L diluted test serum, 50. Mu.L syngeneic Staphylococcus aureus cells (ratio 1 cells/1 bacteria), 50. Mu.L 0.5% w/v rabbit complement and OPA medium to a completion volume of 500. Mu.L/well. Control tubes with staphylococcus aureus in the presence of PMN, test serum or complement alone were included in the assay. The assay tubes were incubated at +37 ℃ for 1 hour with shaking. Dilutions were performed in 3 steps (3 × 1/15 dilutions), 50 μ L of different dilutions were added dropwise 6 times to TSA gel and incubated over 12 hours. Percent bacterial survival was defined at each dilution point, if possible, using the following formula: (number of live bacteria/original inoculum). Times.100.

The data obtained in 2 independent assays are shown in table 2 below (ONS = non-specific opsonophagocytosis):

TABLE 2

The results shown in table 2 indicate that:

1) No bacterial killing was detected from mice immunized with PBS or negative control sera injected with polymer-based formulation alone (data not shown).

2) The PS5-rEPA conjugate elicited anti-PS 5 serum antibodies that showed weak killing activity in the presence of hPMN with a percent bacterial kill of 39% at 1/100 serum dilution. When the serum pool was diluted to 1/500, no more bacterial killing activity was measured.

3) Co-injection of PS5-rEPA with PAA20 did not improve the ability of anti-PS 5 serum antibodies to kill Staphylococcus aureus Lowenstein strains.

4) The adjuvant effect of PAA225000 on bacterial killing was observed in test 1, with a bacterial killing rate of 50% versus 39% for unadjuvanted PS5-rEPA at 1/100 serum dilution and 23% versus 9% for unadjuvanted PS5-rEPA at 1/500 serum dilution.

The general conclusion of this test in relation to staphylococcal antigens is that the adjuvants of the invention show superior efficacy compared to adjuvants with lower molecular weight.

2) Testing of the adjuvant Effect of different polymers on hCMV-gB induced immune responses

The objective of this study was to evaluate the effect of the molecular weight of polyacrylic acid (PAA) polymers on the adjuvant effect. The device was accomplished by using recombinant protein derived from the gB glycoprotein of human cytomegalovirus (hCMV-gB) as a model antigen.

The recombinant protein was produced using a recombinant CHO line transfected with a plasmid designated 0708985pEE14.4 containing a modified gB gene. To facilitate the production of such a recombinant protein by the CHO line, the gB gene of the sequence described in U.S. Pat. No. 5,834,307 was previously altered by deleting the gene portion encoding the transmembrane region of the gB protein corresponding to the amino acid sequence between valine 677 and arginine 752 and introducing 3 point mutations at the cleavage sites. The protein produced by the CHO line, termed gBdTM, corresponds to a truncated gB protein lacking the cleavage site and the transmembrane region.

The gBdTM protein produced in the medium was subsequently purified by chromatography and stored as a stock solution containing >0.2mg/ml gBdTM in phosphate buffer.

PAA with different molecular weights are as follows:

PAA20 and PAA225000 as described and prepared in paragraph "1) assessment of adjuvant effect of the association of PAA of the present invention with staphylococcus aureus antigen compared to 2 PAA of the prior art".

PAA3000 (ref 06568), PAA6000 (ref 06567), PAA50000 (ref 00627) and PAA60000 (ref 18611) are NaPAA and are provided by Polysciences in the form of a dry powder (PAA 6000) or other concentrated solutions.

PAA20 was mixed with water to a concentration of 20mg/ml and stirred at room temperature for 12 hours. The solution was then filtered through a 0.22 μm VDF membrane and stored while maintaining at +4 ℃ as a solution containing 20mg/ml polymer in 150mM NaCl aqueous solution. This solution was then mixed with PBS 10C and sterile water to give a saline solution containing 2mg/ml of polymer.

PAA from Polysciences was diluted with sterile water to a concentration of 20mg/ml, adjusted to pH about 7.4 (except for pH-unadjusted PAA 60000) by using a 2kDa cut-off dialysis cartridge (Thermo Fischer Scientific, cortaboeuf, france) with NaOH or HCl and dialyzed against 150mM NaCl (3 baths in succession). The solution was then filtered through a 0.22 μm pvdf membrane for sterilization. Mw, mn and IP were determined and the polymer was stored at +4 ℃ as a solution containing 20mg/ml of the polymer salt in 150mM NaCl in water.

The molecular weights (Mw and Mn) and PIs of the polymers are shown in Table 3 below:

TABLE 3

	Mw in Da	Mn in Da	Polydispersity index
				PAA3000	Not determined	Not determined	Not determined
PAA6000	9 050	2 940	3.1
				PAA50000	133 460	56 360	2.4
PAA60000	133 760	44 500	3.0
				PAA20	100 700	46 700	2.2
PAA225000	488 550	129 070	3.8

Preparation of a pharmaceutical composition containing Novartis by microfluidization

Squalene emulsionEmulsion of squalene in the same composition to compare the adjuvant activity of different polymers with that of the prior art adjuvants used as reference.

The adjuvanted formulation is prepared by vol/vol mixing of an antigen solution with an adjuvant solution.

The adjuvant dose is 200. Mu.g of polymer per injection dose, or in the case of an emulsion, the final vaccine reagent dose contains 2.5% v/v squalene.

The different formulations tested were as follows (gB corresponds to hCMV-gB: 2. Mu.g in each formulation).

gB alone

-gB + squalene emulsion

-gB+PAA3000

-gB+PAA6000

-gB+PAA50000

-gB+PAA60000

-gB+PAA20(PBS)

-gB+PAA225000

On days 0 and 28, C57BL/6 mice (8-10 per group) were immunized twice with recombinant hCMV-gB antigen (2 μ g/injection) either bound by the IM route or without adjuvant (left quadriceps muscle at a final volume of 50 μ Ι). The assay included a group immunized with hCMV-gB antigen alone as a control.

Blood samples were collected under anesthesia from the submandibular vein at D28 (intermediate bleeding) and bled from all animals by carotid artery dissection at D41. Anesthesia was performed by administering imalgene (1.6 mg ketamine) and xylazine (0.32 mg xylazine) by the intraperitoneal route (IP) in a volume of 150 μ L.

For the D28 humoral response assay, 200 μ L of blood was collected in a vial containing a clot activator and serum separator (Becton Dickinson Microtainer SST, ref 365951). After overnight at +4 ℃, the blood was centrifuged at 10 000rpm for 5 minutes and serum was collected and stored at-20 ℃ until analysis. At D41, 1mL of blood was collected in a vial containing a clot activator and serum separator (BD Vacutainer SST reference 367783). After 12 hours at +4 ℃, the blood was centrifuged at 3000rpm for 20 minutes and serum was collected and stored at-20 ℃ until analysis.

For D41 cellular response assays, spleens were collected from 5 mice per group under sterile conditions. Splenocytes were isolated as follows: freshly collected spleens were dissociated with a Gentlemax dissociator (Miltenyi Biotec), the cell suspension was passed through a cell strainer and washed with RPMI medium. Erythrocytes were lysed using erythrocyte lysate (Red Blood Cell lysis Buffer) (Sigma). After washing, splenocytes were counted and used immediately for cell assay.

Serum neutralization assay:

this technique was used to titrate the functional neutralizing antibodies present in the serum of hCMV-gB immunized animals. Based on the ability of cytomegalovirus to infect MRC5 fibroblasts and ARPE-19 cells (human epithelial cells), sera containing specific functional antibodies against HCMV-gB can inhibit viral infection of cells.

SN50 vs MRC5

Briefly, 1 × 10 ⁴ MRC-5 fibroblasts in DMEM 1% FBS in 96 well flat-bottom plates in 5% CO ₂ The cells were cultured in a cell incubator at 37 ℃ for 1 day. Heat inactivated serum from immunized animals was serially diluted with 1% FBS (fetal bovine serum) in DMEM (Dulbecco's modified Eagle Medium) supplemented with 10% young rabbit complement and incubated with 3.3log CCID50 (cell culture infectious dose 50%)/ml hCMV Towne strain in a cell incubator for vol/vol 1 hour. The serum/virus mixture was then transferred to a monolayer of MRC-5 cells. After 7 days of incubation, the culture supernatant was removed, and the cells were washed 4 times with PBS1C and then fixed with 100. Mu.l of 85% aqueous acetone for 15 minutes at-20 ℃. Plates were washed three times with PBS1C and air dried. Infected cells were detected using a colorimetric reaction. A mixture of two specific hCMV biotinylated antibodies (anti-IE 1CH160 and anti-gB CH177HCMV proteins) was added to the wells at 0.5. Mu.g/ml for 1 hour at room temperature. Plates were washed in PBS 1C/0.05% Tween20 (PBST) and phosphatase alkaline streptavidin was added for 1 hour at room temperature (22 ℃). Plates were washed in PBST and stained with 100. Mu.l chromogen NBT/BCIP for 30 min at room temperature in the dark. After washing, the plates were air dried and scanned using a colorimetric ELISPOT plate reader (Microvision Instruments, evry, france). A deep stain representing cytopathic effect was observed in each wellAnd (4) a core. Serum dilutions were considered neutralized if no dark focus was observed in the corresponding wells. Serum at each dilution was tested in 4 replicates. For each dilution, 50% neutralization (SN 50) was calculated by final square regression. SN50 values (expressed as log 10) defined as the reciprocal of the highest serum dilution, which reduced the number of infected wells by 50%. The average neutralizing antibody titer of each group of mice was calculated.

b. μ PRNT50 vs ARPE-19

Briefly, the day before the ARPE-19 micro-neutralization (MN) assay, 2,5x10 was added ⁴ Individual ARPE-19 cells were distributed in 96-well dark plates. At D0, the sera were heat-inactivated at 56 ℃ for 30 min. Serum samples were serially diluted two-fold in DMEM/F12-FBS, starting from 1/10 to 1/10240 in 96-well plates, and were 5% CO at 37 ℃ with a BADrUL131-Y4HCMV strain of 4.2log FFU/ml ₂ The incubation was performed in a cell incubator for 60 minutes. Then transferring the serum/virus mixture to ARPE-19 cells and 5% CO at 37% ₂ Incubate in cell incubator for 4 days.

At D4, after removal of the culture supernatant, the cells were fixed with 100. Mu.l of 1% formaldehyde in PBS1C for 1 hour at room temperature. The plates were then washed three times with PBS1C and air dried at room temperature, and then analyzed on a Microvision fluorescence plate reader to count infected cells in each well.

As a control, there were two cell control wells (no virus) and six wells on each plate, where cells were infected with half of the virus dilution containing 4,2log FFU/mL. The average of these six wells defined the threshold for serum neutralization, determined as 50% of the specific signal value.

The neutralization endpoint titer is defined as the reciprocal of the last dilution below the calculated 50% specific signal value. The neutralization titer (μ PRNT 50) was defined as each individual serum as the final dilution that induced a 50% reduction in infected cells, i.e. the final dilution induced less cell infection than the calculated 50% specific signal value. Geometric mean neutralizing antibody titers were calculated for each group.

The GMT (neutralizing antibody titer geometric mean) results obtained for each group of immunized mice are shown in figures 2 and 3:

similar properties were observed for serum neutralization assays of MRC5 fibroblasts and ARPE epithelial cells regardless of the group analyzed. No or low neutralizing antibody titers were detected in mice immunized with non-adjuvanted hCMV-gB (GMT = 6).

The polyacrylic acid polymers of the present invention have, to date, achieved the best response.

IgG1 and IgG2c antibody response

Serum IgG1 and IgG2c antibodies against hCMV-gB antigen were titrated by a robotic ELISA assay according to the following method.

Dynex 96-well microplates were coated with 1. Mu.g/well of hCMV-gB in 0.05M carbonate/bicarbonate buffer (pH 9.6) (Sigma) at 4 ℃ for 12 hours. The plates were then blocked with 150. Mu.L/well of PBS Tween-milk (PBS pH7.1,0.05% Tween20,1% (w/v) powdered skim milk (DIFCO)) for 1 hour at 37 ℃. All subsequent incubations were performed in a final volume of 100. Mu.L, followed by 3 washes with PBS pH7.1,0.05% Tween 20. Serial two-fold dilutions of serum samples (starting from 1/100) in PBS-Tween-milk were added to the wells 1/1000). The plates were incubated at 37 ℃ for 90 minutes. After washing, anti-mouse IgG1 or IgG2c peroxidase conjugate (Southern Biotech) diluted 1/2000 in PBS-Tween-milk was added to the wells and the plates were incubated for 90 min at 37 ℃. The plates were further washed and incubated with 100. Mu.L/well of ready-to-use Tetramethylbenzidine (TMB) substrate solution (TEBU) for 30 minutes in the dark at 20 ℃. The reaction was stopped with 100. Mu.L/well of HCl 1M (Prolabo). The Optical Density (OD) was measured at 450nm to 650nm with a plate reader (Spectra Max-molecular devices). IgG antibody titers were calculated using CodUnit software with OD values for the titration curves ranging from 0.2 to 3.0 (reference mouse hyperimmune serum placed on each plate). The IgG titers of this reference, expressed in arbitrary ELISA Units (EU), correspond to log10 giving the reciprocal dilution with an OD of 1.0. The threshold for antibody detection was 10 ELISA units (1.0 log 10). All final titers are expressed as Log10 (Log).

The results are shown in figures 4 and 5:

these results indicate that for IgG1 and IgG2c titers, the adjuvanted hCMV-gB antigen induced an enhanced immune response compared to the unadjuvanted antigen, except for PAA3000 and PAA6000, which had the lowest molecular weight.

It is noteworthy that PAA of the invention is particularly effective in increasing T helper type 1 (Th 1) immune responses, as IgG2c titers are particularly strong in mice immunized with hCMV-gB in combination with PAA of the invention.

Cytokine assay:

splenocytes from immunized mice were isolated immediately after sacrifice on day 41 to 2.5x10 per well ⁵ Individual cells were seeded in 96-well plates and incubated with hCMV-gB (5 μ g/well), concanavalin a (0.25 μ g/well; positive control) or culture medium. Used alone (RPMI-GSP. Beta. -10% FCS; background). After 6 days of incubation, secretion of IL5 and IFN γ cytokines was measured using the CBA Flex set Kit. Results are expressed as cytokine concentrations in pg/ml (geometric mean of each group). The threshold for positive cytokine detection for IL-5 was 5pg/ml and for IFN γ was 2.5pg/ml.

The results are shown in fig. 6 and 7:

these results indicate that with emulsion adjuvants, the level of IL5 is high, while the level of IFN γ is low. Significantly, the polyacrylic acid adjuvants of the invention induced low levels of IL5, but high levels of IFN γ, which is indicative of a Th 1-biased immune response. This result is consistent with the immunoglobulin subtyping results.

3) Comparison of diafiltered PAA of the present invention with its raw material before diafiltration

In this test, it was demonstrated that diafiltration did not affect the adjuvant properties of the polymer.

Testing different formulations containing 0.08mg/mL of hCMV-gB antigen were obtained as described in paragraph "2) adjuvant effect test of different polymers on hCMV-gB induced immune responses".

From sodium salts of polyacrylic acid polymers supplied by Polysciences, two different methods were applied (undiffiltrated preparation and diafiltered preparation).

For the undiluted formulation, the product from Polysciences was briefly diluted with PBS and sterile filtered through a membrane with a 0.2 μm cut-off. The concentration of PAA salt was 17.4mg/mL. The Mw, IP and Mark-Houwink slopes were determined.

For the diafiltration formulation, the product from Polysciences was processed according to the following protocol:

a) The aqueous solution supplied by Polysciences was mixed with PBS1C pH7.4 with stirring during 15 minutes to obtain a PBS solution containing 14mg/ml of polymer,

b) Diafiltering the solution obtained in step a) against 5 volumes of PBS, wherein the membrane has a cut-off of 50kDa,

c) The product obtained in step b) was filtered through a 0.2 μm sterile filter.

The resulting solution contained 15.9mg/mL of the PAA salt at a pH of 7.3. The Mw, IP and Mark-Houwink slopes were determined.

The Mw, IP and Mark-Houwink slopes obtained before and after the diafiltration obtained are shown in Table 4

TABLE 4

NaPAA	Mw(Da)	IP	Mark-Houwink slope
				Diafiltration (Perfect Inflation)	522,030	1.6	0.9
Without percolation	433,417	2.6	0.9

The molecular weights Mw and IP of the polymer before and after diafiltration are consistent with the following facts: monomers and small oligomers, especially those less than 2000 daltons, have been eliminated by the diafiltration step.

It is also important to note that the Mw, as determined by GPC (gel permeation chromatography), published by Polysciences is 887,000Da, which is quite different from the Mw measured before diafiltration.

From these two different formulations, an immune preparation was prepared by mixing one of the polymer formulations with a gB solution in the appropriate ratio to obtain 50 μ Ι doses, each dose containing 2 μ g of gB, and:

25, 50, 100 or 200 μ g of polymer salt from the diafiltration formulation,

or 25, 50, 100 or 200 μ g of polymer salt from the undiluted formulation.

On day 0 and 21, 7-week-old C57BL/6J mice were immunized twice with one of the prepared preparations by intramuscular route. Each formulation was tested in a group of 5 mice. As a control, a group of 5 mice received the antigen alone.

At 2 weeks after the last immunization (day 35), the cells (IFN γ and IL 5) and humoral responses (IgG antibody subclass, serum neutralizing antibodies) of the immunized mice were monitored in the same manner as described in paragraph "test of adjuvant effect of different polymers on hCMV-gB induced immune response".

The results show that, as in the previous experiments, the adjuvant of the invention induced a strong Th-1 immune response, together with a strong virus-neutralizing antibody titer, and that there was no significant difference between mice immunized with the diafiltered formulation and those immunized with the non-diafiltered formulation.

4) Comparative stability study of diafiltered PAA with PAA feedstock

To examine the stability of the PAA adjuvant of the present invention, a study was conducted which analyzed the change in molecular weight of the polymer salt in an accelerated aging test.

For this test, the undiluted formulation and the diafiltered formulation were used. Purification was performed by diafiltration using a50 kDa cut-off membrane and the resulting diafiltration preparation contained 8mg/mL of a PAA polymer salt with a molecular weight of 443553Da in PBS 1C.

It is present in the sodium persulfate in an amount of less than 0.0007% (w/w) dry polymer; the sodium acrylate content was determined to be 0.0011% of dry polymer.

The formulation was filled into glass vials and maintained at +5 ℃, +25 ℃ or +37 ℃.

The analysis was performed at 5 ℃ for 24 months, 25 ℃ for 9 months and 37 ℃ for 3 months. The results obtained during this stability study are shown in table 5 below:

TABLE 5

These results show that there is no significant decrease in Mw after 24 months of storage at 5 ℃, after 9 months of storage at 25 ℃ and after 3 months of storage at 37 ℃ and that the polydispersity index of the polymer remains unchanged.

This is in contrast to the results obtained using a composition corresponding to an aqueous solution containing 10% w/w of undiluted, comparable PAA salt containing sodium persulfate in a concentration of 0.42% w/w relative to the dry weight composition. The initial Mw of the polymer salt was determined to be 470,269Da, while the supplier's published Mw as determined by GPC was 351,100Da.

The results obtained during the stability study of the non-dialyzed polymer solution are shown in table 6 below and show a decrease in molecular weight over time, particularly after storage at 25 ℃ and 37 ℃.

TABLE 6

5) Shows the persulfate contentTesting for detrimental effects of

The sodium salt of the polyacrylic acid polymer (supplied by Polysciences) was sterilized in an autoclave at a temperature of 121 ℃ for 15 minutes. The concentration of the PAA salt in the solution was 101.8mg/g. Table 7 below shows the Mw, IV (intrinsic viscosity) and mark-houwink slope of the PAA salt before and after autoclaving.

TABLE 7

NaPAA	Mw(Da)	IV(dl/g)	Mark-Houwink slope
				Before autoclaving	404784	3.5	0.9
After autoclaving	188782	1.9	0.9

It is clear that the treatment in the autoclave leads to a sharp drop in Mw and IV. Subsequent studies were conducted to investigate parameters that may lead to degradation of the polymer under heat. This study shows that purification of the polymer salt, in particular by diafiltration, is capable of stabilizing the polymer when sterilized by autoclaving.

The mixture of NaPAA polymer and persulfate was exposed to heat to reproduce the conditions of autoclaving: the composition was characterized for NaPAAMw and persulfate content before and after 15 minutes incubation at 120 ℃. Table 8 details the compositional characteristics in terms of NaPAA Mw and persulfate content.

TABLE 8

These results show that the presence of persulfate leads to a decrease in Mw after heat treatment. In contrast, PAA with very weak persulfate content has a very stable Mw.

In summary, the thermal stability of PAA solutions may be directly related to their persulfate content. The diafiltration step introduced in the process of the invention removes persulfate impurities from the PAA solution and provides a heat stable PAA solution compatible with sterilization by autoclaving.

Example 3-PAA supports a strong response and reduces the antigen payload

The need for more extensive protection of livestock pathogens requires the addition of antigens to existing vaccine formulations (e.g.

Products, which typically comprise inactivated toxin and/or bacterin plus aluminum hydroxide adjuvant) or a single preparation of a single antigen vaccine. As it is more commercially desirable to provide combination vaccines, applicants concluded that they could add new antigens while still maintaining the existing dose volumes, provided they could identify more effective adjuvants with good safety.

Importantly, as is well known to those skilled in the art of vaccines and immunology, one cannot predict in advance whether a molecular species will function as a safe and effective immunogenic adjuvant. Furthermore, even after the adjuvant properties of the new molecules have been established, the skilled person cannot reasonably expect the success of applying the new adjuvants to different situations due to the unpredictable nature of these techniques. Variables that affect whether the new formulation functions as a safe and protective vaccine include, but are not limited to: 1) Adjuvant type/presence; 2) Antigen type/nature (peptide, nucleic acid, virus killed, bacterin killed etc.); 3) The route of administration; 4) Target pathogen type/strain; and 5) the target species to be vaccinated. Thus, the safety and efficacy of the new vaccine adjuvants must be demonstrated in at least several combinations of these variables before any meaningful conclusions can be drawn as to their general applicability.

For the case of the present invention, where the antigen type comprises mainly inactivated bacteria, the applicants conclude that the final new, more effective and dose-retaining adjuvant must be lipase resistant. Unfortunately, compounds that render the formulation lipase resistant often result in undesirable application site reactions. Therefore, there is a need to have a good balance between vaccine safety, efficacy and stability.

Preparative experiments

Vaccines (Merial Brazil-Clostridium perfringens B/C, clostridium perfringens D, clostridium septicum, clostridium nokohlii, clostridium tetani) and tested on mice, guinea pigs, pigs and rabbits. By way of example, guinea pig study design and results are shown in tables 9 and 10, respectively.

TABLE 9 dose reduction regimen Using PAA225000 polyacrylic acid adjuvant

	(% standard vaccine)	Pre-adsorption of antigen on aluminium hydroxide	Dosage PAA (mg/dose)
				Basic vaccine	1	Is that	0
A	0.2	Is that	1
				B	0.2	Is that	2
C	0.2	Is that	3
				D	0.2	Is that	4
E	0.2	Whether or not	4

Guinea pigs, mice and rabbits are safe and, as shown in table 10, function well, particularly for formulations lacking aluminum hydroxide (see, e.g., novyi, septicum and sordelii columns; compare E with D). These results are very surprising, as it can reasonably be expected that the combination of aluminium hydroxide and the new polymer adjuvant (group E) will outperform the new polymer-only formulation (group D).

Example 4-PAA supports a protective response when formulated using a traditional inactivated or recombinant vaccine

Inactivated rabies vaccine was prepared according to table 11 and the results are shown in figure 8. All adjuvants tested were apparently safe for use in dogs, and all induced seroconversion at day 7, maintaining positive titers for up to 70 days after vaccination. PAA225000 is the most effective adjuvant for improving the short-term immunogenicity of inactivated rabies.

TABLE 11 inactivated rabies vaccine formulation (of Merial

+ different adjuvants

* AF03 is an alternative adjuvant based on squalene emulsions produced using phase inversion (see j. Pharm. Sciences, vol 101, no. 12, 2012, and incorporated herein by reference in its entirety).

* Preparing a squalene emulsion using high pressure homogenization to form an oil-in-water emulsion.

Canarypox-vectored influenza vaccine formulations were prepared and tested in 5 groups, each group containing 7 dogs (table 12), with the results shown in figure 10. High levels of IFN γ -producing cells were detected in all groups D14, D27 and D41. Similar to the trends observed with the classical inactivated vaccine formulations described above, canarypox vector influenza antigen is apparently better adjuvanted by a higher MW PAA (i.e. PAA 225000). The vCP2242 is fully described and implemented by US7,425,336) (Merial), and is incorporated herein by reference in its entirety. Briefly, however, vCP2242 is a recombinant ALVAC containing a codon-optimized HA gene from H3N8 Equine Influenza Virus (EIV) inserted into the ALVAC C5 locus. Applicants assert that the results disclosed herein support the general conclusion that recombinant canarypox vectors are compatible with and well adjuvanted by the non-crosslinked PAA of the present disclosure (see, e.g., fig. 9).

TABLE 12 Canine influenza vaccine formulation with canarypox as a carrier

Example 5 non-crosslinked PAA is an effective adjuvant for equine vaccines

Canarypox-vectored equine influenza + tetanus toxin study formulations were administered to equine animals according to table 13. As shown below, and in fig. 11-13, PAA strongly helped two unrelated antigens to elicit a protective immune response in equine animals.

TABLE 13 equine influenza + tetanus formulation with canarypox as carrier

ADVAX ^TM The adjuvant is derived from insulin (vaccine: 8.3.2012; 30 (36): 5373-81; see also US2014/0314739, vaxine Pty Ltd.). ADVAX1 is a preservative-free sterile suspension of delta insulin microparticles at 20mg/ml in bicarbonate buffer, while ADVAX2 also includes 10. Mu.g CpG dinucleotide/1 mg delta insulin.

Results by D14, the protective titer was > 0.05IU/ml for all PAA225000 animals. At D14, 6 of the 8 animals from the carbomer adjuvanted vaccine group were still <0.05IU/ml. Thus, the PAA225000 adjuvanted vaccine formulation produced significantly better seroconversion than the carbomer adjuvanted immune formulation. By D49 (after the second vaccination), titers in PAA225000 group were significantly and effectively high in all animals.

Example 6-PAA is an effective adjuvant for porcine vaccines

"SpaA" antigen porcine research it is contemplated that "SpaA" refers to a "surface protective antigen of Erysipelothrix rhusiopathiae, which is a pathogen that infects porcine and other animals, including canines. Erysipelothrix rhusiopathiae is a gram-positive, catalase-negative, rod-shaped, non-sporulating, non-acid-fast, non-motile bacterium. In porcine animals, erysipelothrix rhusiopathiae causes "mild swine erysipelas". "TS6" refers to an oil-in-water emulsion, as described, for example, in U.S. Pat. No. 7,371,395 (Merial). TS6 was formulated by the following steps: add about one (1) part of an aqueous component comprising an antigen to about two (2) parts of an oily component, and then emulsify the two components to form a final emulsion.

TABLE 14 combination therapy definitions

Group of	Number of	Treatment of
			G1	7	SpaA antigen (150) ¹ μ g/dose) + TS6
G2
		7	SpaA antigen (100. Mu.g/dose) + PAA60000
G3
		7	SpaA antigen (100. Mu.g/dose) + PAA225000
G4
		7	SpaA-FlaB-His antigen (196) ² μ g/dose) + PBS
G5
		7	SpaA-FlaB-His antigen (196) ² μ g/dose) + PAA225000
G6
		7	PBS

¹ 150 μ g of SpaA in 2/3 of the treatment volume (tt) means 100 μ g of SpaA per 1/1 of the volume tt.

¹ 196 μ g of SpaA fusion protein corresponds to 100 μ g of orphan SpaA protein.

Thus, an effective amount of SpaA delivered to each group is 100 μ g.

While G1 produced the best results, it is noteworthy that the non-antigenic component occupied most of the dose volume of the oil-in-water emulsion (as described above, the ratio of oily component to aqueous antigenic component was 2.

The invention will now be summarized in the following numbered paragraphs:

1. a pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use as an adjuvant in a vaccine composition, characterised in that said polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 350-650 kDa.

2. The pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to paragraph 1, characterized in that the polyacrylic acid polymer salt consists entirely of units corresponding to the acrylate salt or consists entirely of units corresponding to the free acid form of acrylic acid and units corresponding to the acrylate salt.

3. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to

paragraph

1 or 2, characterized in that it comprises less than 0.005% w/w oxidizing agent, based on the total dry weight of the polyacrylic acid polymer salt.

4. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to any of the preceding paragraphs, characterised in that it comprises less than 0.001% w/w of oxidising agent, based on the total dry weight of the polyacrylic acid polymer salt.

5. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to any of paragraphs 1-3, characterized in that it comprises less than 0.005% w/w of the persulfate salt, based on the total dry weight of the polyacrylic acid polymer salt.

6. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to any of the preceding paragraphs, characterised in that it comprises less than 0.001% w/w of a persulfate salt, based on the total dry weight of the polyacrylic acid polymer salt.

7. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to any of the preceding paragraphs, characterized in that the polyacrylic acid polymer is reacted with Na ⁺ A salt.

8. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for its use according to any of the preceding paragraphs, characterized in that the polyacrylic acid polymer salt has a polydispersity index of less than or equal to 4, preferably less than or equal to 2.5.

9. The pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for its use according to any of the preceding paragraphs, characterized in that the polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 380 to 620kDa and a polydispersity index of less than or equal to 4; or has a weight-average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 4; or has a weight-average molecular weight Mw in the range from 380 to 620kDa and a polydispersity index of less than or equal to 2.5; or has a weight-average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 2.

10. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to any of the preceding paragraphs, characterized in that the polyacrylic acid polymer salt has a mark-houwink slope of greater than or equal to 0.7.

11. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for its use according to any of the preceding paragraphs, characterized in that it comprises less than 0.005% w/w acrylic acid monomer in free acid form or in salt form, based on the total dry weight of the polyacrylic acid polymer salt.

12. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for its use according to any of the preceding paragraphs, characterised in that it is in the form of a liquid formulation having a pH in the range 5.5-8.0.

13. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer in the form of a salt for its use according to paragraph 10, characterized in that it is in the form of an aqueous buffered solution, in particular using a phosphate buffer or TRIS, hepes, histidine or citrate buffer.

14. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for its use according to any of the preceding paragraphs, characterised in that it is diafiltered.

15. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for its use according to any of the preceding paragraphs, characterised in that it is sterilised.

16. A pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer for use according to any of the preceding paragraphs, characterised in that it is for use in enhancing a Th1 immune response obtained with the vaccine composition.

17. A vaccine composition comprising at least one vaccine agent and a pharmaceutically acceptable salt of a polyacrylic acid polymer according to any of paragraphs 1-16 as an adjuvant.

18. The vaccine composition according to paragraph 17, characterized in that it comprises 0.1-8mg of the pharmaceutically acceptable salt of polyacrylic acid polymer per dose, preferably 0.1-4mg, and more preferably 0.1-2mg.

19. A vaccine composition according to paragraphs 17 or 18, characterized in that the at least one vaccine agent is an antigen or a vector expressing an antigen, such as a viral vector or a nucleic acid.

20. Vaccine composition according to paragraph 18, characterized in that the antigen is a bacterial antigen derived from Clostridium tetani (Clostridium tetani), clostridium diphtheriae (Clostridium diphtheriae), bordetella pertussis (Bordetella pertussis), haemophilus influenzae type B (Haemophilus influenzae type B), streptococcus pneumoniae (Streptococcus pneoniae), neisseria meningitidis (Neisseria meningitidis), shigella (Shigella sp), salmonella typhi (Salmonella typhi), staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), mycobacterium tuberculosis (Mycobacterium tuberculosis), chlamydia trachomatis or Chlamydia pneumoniae (Chlamydia trachomatis or Streptococcus sp) or Streptococcus (Streptococcus sp); or viral antigens derived from hepatitis A, B or C virus (hepatis A, B or Cvirus), influenza virus (influenza virus), respiratory syncytial virus (respiratory syncytial virus), rhinovirus (rhinovirus), west Nile virus (West Nile virus), rabies virus (rabies virus), poliovirus (poliovirus), HIV virus (HIV virus), dengue virus (dengue virus), japanese encephalitis virus (Japanese encephalititis virus), yellow fever virus (yellow fever virus), cytomegalovirus (cytomegalovirus) or herpes virus (herpes virus); or is a parasite antigen from Plasmodium sp, leishmania sp or Schistosoma sp; or a tumor antigen.

21. Vaccine composition according to any of paragraphs 17-19, characterized in that the at least one vaccine agent is an antigen or a vector encoding an antigen, such as a recombinant virus or a nucleic acid, said antigen being derived from staphylococcus aureus or cytomegalovirus.

22. A vaccine composition according to any of paragraphs 17 to 21, characterized in that it is in liquid form having a pH in the range of 6.0 to 8.0.

23. A vaccine composition according to paragraph 21, characterized in that it is in the form of an aqueous buffered solution, in particular using a phosphate buffer or TRIS, hepes, histidine or citrate buffer.

24. A vaccine composition according to any of paragraphs 17-23 for use in enhancing an immune response in an individual, particularly a human, enhancing the obtained Th1 immune response and/or balancing the obtained Th1 and Th2 immune responses.

25. A process for preparing a pharmaceutically acceptable salt of a polyacrylic acid polymer according to any of paragraphs 1 to 16, the process comprising the sequential steps of:

a) A solution of a polyacrylic acid polymer is obtained,

c) The solution of purified polyacrylic acid polymer is sterilized.

26. The preparation process according to paragraph 25, characterized in that the polyacrylic acid polymer of the solution of step a) has a weight average molecular weight Mw in the range of 300-550 kDa.

27. The preparation method according to paragraph 25 or 26, characterized in that the purification is carried out by dialysis, diafiltration, ultrafiltration or size exclusion chromatography.

28. The preparation process according to paragraph 27, characterized in that the purification is carried out by diafiltration using a membrane with a cut-off of 1-80kDa, preferably 2-50 kDa.

29. The preparation process according to any of paragraphs 25 to 28, characterized in that the purification is carried out under conditions allowing to obtain a polyacrylic acid polymer in the form of a solution having:

-less than 0.005%, preferably less than 0.001% w/w, of oxidizing agent, based on the total dry weight of the polyacrylic acid polymer obtained after purification; and/or less than 0.005%, preferably less than 0.001% w/w of persulfate, based on the total dry weight of the polyacrylic acid polymer obtained after purification,

-less than 0.005% w/w of acrylic acid monomers in free acid form or in salt form, based on the total dry weight of the polyacrylic acid polymer obtained after purification,

-for polyacrylic acid polymer salts: a weight average molecular weight Mw in the range from 380 to 620kDa and a polydispersity index of less than or equal to 4; or a weight average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 4; or a weight average molecular weight Mw in the range from 380 to 620kDa and a polydispersity index of less than or equal to 2.5; or a weight average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 2.

30. The production method according to any one of paragraphs 25 to 29, characterized in that the sterilization is performed in an autoclave.

31. The preparation method according to any of paragraphs 25-30, characterized in that the purification and sterilization are performed on a solution of a pharmaceutically acceptable salt of the polyacrylic acid polymer.

32. The preparation method according to any of paragraphs 25-31, characterized in that the purification is performed on a solution comprising 2-50mg/mL of a pharmaceutically acceptable salt of the polyacrylic acid polymer.

33. A method for storing a solution of a polyacrylic acid polymer salt according to any of paragraphs 1 to 16, the method comprising a preparation method according to any of paragraphs 25 to 32, followed by a storage step of the resulting pharmaceutically acceptable salt of the polyacrylic acid polymer in solution.

34. Storage method according to paragraph 33, characterized in that the storage step lasts at least 1 day and at most 2 years.

35. Storage method according to paragraph 33 or 34, characterized in that the storage step is performed by placing a solution of a polyacrylic acid polymer salt in a container at a temperature of 0-30 ℃, preferably 2-8 ℃.

36. Storage method according to any of paragraphs 33-35, characterized in that the solution of polyacrylic acid polymer salt is kept protected from light during storage.

37. An immunological or vaccine composition comprising a therapeutically effective amount of an antigenic component, a pharmaceutically or veterinarily acceptable carrier, and an adjuvant comprising or consisting essentially of a non-crosslinked polyacrylic acid (PAA) polymer having a Mw of about 350kDa to about 650kDa and a polydispersity index of less than about 4 or less than about 2.

38. The composition according to paragraph 35, wherein the PAA has a Mw of about 400kDa to about 600 kDa.

39. The composition according to paragraph 36, wherein the PAA has a Mw of about 400kDa to about 500 kDa.

40. An immunization or vaccine composition according to paragraph 35 wherein the antigenic component comprises an attenuated recombinant viral vector, a naturally or genetically engineered attenuated live virus or microorganism, an inactivated virus or microorganism, a coccidia precocious, a protein subunit, a unicellular parasite, a multicellular parasite or any combination of the foregoing.

41. An immunization or vaccine composition according to paragraph 35 wherein the antigenic component comprises: an Eimeria sp or antigen thereof, an Escherichia coli (e.coli) or antigen thereof, a Mycoplasma hyopneumoniae (m.hyo), a Bovine Diarrhea Virus (BDV) antigen, a recombinant canarypox vector containing and capable of expressing at least one protective immunogen in vivo, an inactivated full length rabies glycoprotein, a erysiphe sp, a erysiphe rhusiopathiae, a surface protective antigen from erysiphe rhusiopathiae (SpaA), a spa fusion protein comprising at least a portion of at least one additional immunogen, a spa-FlaB fusion protein, a spa-FlaB-His fusion protein, a Clostridium perfringens (C) B/C toxin, a Clostridium capsular (C) toxin, clostridium perfringens (c.clostridium tetani.c.toxin, clostridium perfringens.

42. An immunization or vaccine composition according to paragraph 39, wherein the antigenic component comprises or consists of inactivated full length rabies glycoprotein.

43. The immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises or consists of Clostridium perfringens B/C toxin, clostridium perfringens D toxin, clostridium septicum toxin, clostridium novyi toxin, clostridium tetani toxin, or a combination thereof.

44. An immunization or vaccine composition according to paragraph 39, wherein the antigenic component comprises Clostridium perfringens B/C toxin, clostridium perfringens D toxin, clostridium septicum toxin, clostridium nokohlii toxin, and Clostridium tetani toxin.

45. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises a SpaA antigen or a fusion protein comprising a SpaA antigen.

46. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises an attenuated fowlpox virus or a DNA plasmid which contains and is capable of expressing an influenza gene in vivo.

47. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises an attenuated fowlpox virus or a DNA plasmid which contains and is capable of expressing the rabies glycoprotein gene in vivo.

48. A method of treating a bovine animal against an infection caused by a bacterium, the method comprising administering to the bovine animal a vaccine composition according to paragraph 41.

49. A method of treating a canine or equine animal against an infection caused by influenza, the method comprising administering to the canine or equine animal a vaccine composition according to paragraph 44.

50. A method of treating a canine against infection by rabies virus, the method comprising administering to the canine a vaccine composition according to paragraph 44.

51. An avian coccidiosis vaccine for in ovo administration, said vaccine comprising:

(a) An adjuvant comprising non-crosslinked PAA having an average Mw of about 350kDa to about 650 kDa; and

(b) A protozoan antigen selected from the group consisting of: (1) one or more recombinantly expressed proteins; (2) One or more proteins or other macromolecules isolated from the protozoa by conventional means; (3) A whole cell extract or preparation from said protozoa; and (4) inactivated, live or live precocious coccidia selected from the group consisting of: eimeria acervulina (Eimeria (e), eimeria acervulina), eimeria adenoids (e.adeneides), eimeria brunetti (e.brunetti), eimeria colchica (e.colchici), eimeria curvata (e.currvata), eimeria dispersa (e.dispersa), eimeria dodecaalis (e.duodenalis), eimeria castellata (e.fratercula), eimeria gabonensis (e.galopovaes), eimeria innocua (e.incocua), eimeria praecox (e.praecox), eimeria maxima (e.maxima), eimeria perviviparii (e.melela), eimeria perreas and Eimeria mitis (e.mitis), eimeria perlecanii (e.mitis), eimeria acervulina (e.mitis), eimeria acervulineria acervulina (e.mitis), eimeria acervulina (e.mitis).

52. A method of treating a bovine animal against an infection caused by E.coli or Mycoplasma hyopneumoniae, the method comprising administering to the bovine animal a vaccine composition according to paragraph 39, wherein the antigenic component comprises E.coli or Mycoplasma hyopneumoniae.

53. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises Mycoplasma hyopneumoniae.

54. A method of treating a porcine animal against an infection caused by Mycoplasma hyopneumoniae, the method comprising administering to the porcine animal a vaccine composition according to paragraph 51.

55. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises FIV.

56. A method of treating a feline against an infection caused by FIV, the method comprising administering to the feline a vaccine composition according to paragraph 53.

57. A vaccine composition according to paragraph 39, wherein the antigenic component comprises a cancer antigen.

58. A method of treating a subject against cancer, the method comprising administering to the subject a vaccine composition according to paragraph 55.

59. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises Canine Coronavirus (CCV).

60. A method of treating canines against infection by CCV, the method comprising administering to the canines a vaccine composition according to paragraph 57.

61. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises bovine rotavirus.

62. A method of treating a bovine animal against infection by bovine rotavirus, the method comprising administering to the bovine animal a vaccine composition according to paragraph 59.

63. An immunization or vaccine composition according to paragraph 39 wherein the antigenic component comprises Canine Influenza Virus (CIV).

64. A method of treating a canine against infection by CIV, the method comprising administering to the canine a vaccine composition according to paragraph 61.

***

As disclosed herein, applicants have for the first time discovered that certain molecular weight ranges of non-crosslinked (i.e., linear and branched) polyacrylic acid (PAA) polymers are particularly suitable for aiding the action of immunogenic antigens, as well as eliciting antigen-independent immune responses. Importantly, applicants have surprisingly found that these non-crosslinked PAA adjuvants are widely used for many different antigen types: an attenuated recombinant viral vector; classical inactivated rabies glycoprotein; a SpaA peptide subunit; and bacterial toxins. In addition, the disclosed PAA adjuvants work well in a variety of animal types. Thus, applicants believe that the disclosed non-crosslinked PAA represents a novel and inventive "universal adjuvant".

Claims

1. Vaccine adjuvant which is a pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer, characterized in that said polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 350-650kDa and a polydispersity index of less than or equal to 4; and comprises less than 0.005% w/w of an oxidizing agent, based on the total dry weight of the polyacrylic acid polymer salt, and wherein the polyacrylic acid polymer consists entirely of acrylic acid units.

2. Vaccine adjuvant according to claim 1, characterized in that the polyacrylic acid polymer salt consists entirely of acrylic acid units being an acrylate salt or consists entirely of acrylic acid units being the free acid form of acrylic acid and acrylic acid units being an acrylate salt.

3. A vaccine adjuvant according to claim 1 or 2, characterised in that it comprises less than 0.001% w/w of oxidising agent, based on the total dry weight of the polyacrylic acid polymer salt.

4. A vaccine adjuvant according to claim 1 or 2, characterised in that it comprises less than 0.005% w/w of persulfate salt, based on the total dry weight of the polyacrylic acid polymer salt.

5. A vaccine adjuvant according to claim 1 or 2, characterised in that it comprises less than 0.001% w/w of persulfate salt, based on the total dry weight of the polyacrylic acid polymer salt.

6. Vaccine adjuvant according to claim 1 or 2, characterized in that the polyacrylic acid polymer is reacted with Na ⁺ A salt.

7. Vaccine adjuvant according to claim 1 or 2, characterized in that the polyacrylic acid polymer salt has a polydispersity index of less than or equal to 2.5.

8. A vaccine adjuvant according to claim 1 or 2 characterised in that the polyacrylic acid polymer salt has a weight average molecular weight Mw in the range 380 to 620kDa and a polydispersity index of less than or equal to 4; or has a weight-average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 4; or has a weight-average molecular weight Mw in the range from 380 to 620kDa and a polydispersity index of less than or equal to 2.5; or has a weight-average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 2.

9. Vaccine adjuvant according to claim 1 or 2, characterized in that the polyacrylic acid polymer salt has a Mark-Houwink slope of greater than or equal to 0.7.

10. Vaccine adjuvant according to claim 1 or 2, characterized in that it comprises less than 0.005% w/w acrylic acid monomer in free acid form or in salt form, based on the total dry weight of the polyacrylic acid polymer salt.

11. A vaccine adjuvant according to claim 1 or 2, characterised in that it is in the form of a liquid formulation having a pH in the range of 5.5-8.0.

12. A vaccine adjuvant according to claim 11 characterised in that it is in the form of an aqueous buffered solution.

13. Vaccine adjuvant according to claim 12, characterized in that the buffered aqueous solution is in the form of phosphate buffer or TRIS, hepes, histidine or citrate buffer.

14. A vaccine adjuvant according to claim 1 or 2, characterised in that it is diafiltered.

15. A vaccine adjuvant according to claim 1 or 2, characterised in that it is sterilised.

16. A vaccine adjuvant according to claim 1 or 2, characterized in that it is used to enhance the Th1 immune response obtained with said vaccine composition.

17. A vaccine composition comprising at least one vaccine agent and a vaccine adjuvant according to any one of claims 1-16.

18. Vaccine composition according to claim 17, characterised in that it comprises 0.1-8mg, or 0.1-4mg, or 0.1-2mg of a pharmaceutically acceptable salt of a polyacrylic acid polymer per dose.

19. Vaccine composition according to claim 17 or 18, characterized in that the at least one vaccine agent is an antigen or a vector expressing an antigen.

20. Vaccine composition according to claim 19, characterized in that the antigen is an antigen derived from Clostridium tetani (Clostridium tetani), clostridium diphtheriae (Clostridium dithermophile), bordetella pertussis (Bordetella pertussis), haemophilus influenzae type B (Haemophilus influenzae type B), streptococcus pneumoniae (Streptococcus pneumoniae), neisseria meningitidis (Neisseria meningitidis), shigella (Shigella sp), salmonella typhi (Salmonella typhi), staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), mycobacterium tuberculosis (Mycobacterium tuberculosis), chlamydia trachomatis, or Chlamydia pneumoniae (Chlamydia pneumoniae or pneumococcus) or Streptococcus (Streptococcus sp); or viral antigens derived from hepatitis A, B or C virus (hepatitis A, B or C virus), influenza virus (influenza virus), respiratory syncytial virus (respiratory syncytial virus), rhinovirus (rhinovirus), west Nile virus (West Nile virus), rabies virus (rabes virus), poliovirus (poliovirus), HIV virus (HIV virus), dengue virus (dengue virus), japanese encephalitis virus (Japanese encephalitism virus), yellow fever virus (yellow fever virus), cytomegalovirus (cytomegavirus) or herpes virus (herpes virus); or is a parasite antigen derived from Plasmodium sp, leishmania sp or Schistosoma sp; or a tumor antigen.

21. Vaccine composition according to claim 17 or 18, characterised in that the at least one vaccine agent is an antigen or a vector encoding an antigen derived from staphylococcus aureus or cytomegalovirus.

22. Vaccine composition according to claim 17 or 18, characterized in that it is in liquid form having a pH in the range of 6.0-8.0.

23. Vaccine composition according to claim 22, characterized in that it is in the form of a buffered aqueous solution.

24. Vaccine composition according to claim 23, characterized in that the aqueous buffer solution is in the form of a phosphate buffer or TRIS, hepes, histidine or citrate buffer.

25. A process for the preparation of a vaccine adjuvant according to any one of claims 1-16, the process comprising the following successive steps:

a) A solution of a polyacrylic acid polymer is obtained,

c) The solution of purified polyacrylic acid polymer is sterilized.

26. Preparation process according to claim 25, characterized in that the polyacrylic acid polymer of the solution of step a) has a weight average molecular weight Mw in the range of 300 to 550 kDa.

27. Preparation process according to claim 25 or 26, characterized in that the purification is carried out by dialysis, diafiltration, ultrafiltration or size exclusion chromatography.

28. Preparation process according to claim 27, characterized in that the purification is carried out by diafiltration using a membrane with a cut-off of 1-80kDa or 2-50 kDa.

29. Preparation process according to claim 25 or 26, characterized in that the purification is carried out under conditions which allow to obtain the polyacrylic acid polymer in the form of a solution having:

-less than 0.005%, or less than 0.001% w/w, based on the total dry weight of the polyacrylic acid polymer obtained after purification, of an oxidizing agent; and/or less than 0.005%, or less than 0.001% w/w of a persulfate, based on the total dry weight of the polyacrylic acid polymer obtained after purification,

-for polyacrylic acid polymer salts: a weight average molecular weight Mw in the range from 380 to 620kDa and a polydispersity index of less than or equal to 4; or a weight average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 4; or a weight-average molecular weight Mw ranging from 380 to 620kDa and a polydispersity index lower than or equal to 2.5; or a weight-average molecular weight Mw in the range from 400 to 600kDa and a polydispersity index of less than or equal to 2.

30. Preparation method according to claim 25 or 26, characterized in that the sterilization is carried out in an autoclave.

31. The process according to claim 25 or 26, characterized in that the purification and sterilization are carried out on a solution of a pharmaceutically acceptable salt of the polyacrylic acid polymer.

32. The method for preparing according to claim 25 or 26, characterized in that the purification is performed on a solution comprising 2-50mg/mL of a pharmaceutically acceptable salt of a polyacrylic acid polymer.

33. A process for storing a solution of a vaccine adjuvant according to any one of claims 1 to 16, the process comprising a preparation process according to any one of claims 25 to 32, followed by a storage step of the resulting pharmaceutically acceptable salt of the polyacrylic acid polymer in solution.

34. Storage method according to claim 33, characterized in that the storage step lasts at least 1 day and at most 2 years.

35. The storage method according to claim 33 or 34, characterized in that the storage step is performed by placing the solution of polyacrylic acid polymer salt in a container at a temperature of 0-30 ℃, or 2-8 ℃.

36. Storage method according to claim 33 or 34, characterized in that the solution of polyacrylic acid polymer salt is kept protected from light during storage.

37. An immunological or vaccine composition comprising a therapeutically effective amount of an antigenic component, a pharmaceutically or veterinarily acceptable carrier, and an adjuvant comprising or consisting of a non-crosslinked polyacrylic acid (PAA) polymer salt having a Mw of from 350kDa to 650kDa and a polydispersity index of less than 4 or less than 2; and comprises less than 0.005% w/w of an oxidizing agent, based on the total dry weight of the polyacrylic acid polymer salt, and wherein the polyacrylic acid polymer consists entirely of acrylic acid units.

38. The composition according to claim 37, wherein the polyacrylic acid (PAA) polymer salt has a Mw of 400kDa to 600 kDa.

39. The composition according to claim 38, wherein the polyacrylic acid (PAA) polymer salt has a Mw of 400kDa to 500 kDa.

40. The immunization or vaccine composition of claim 37 wherein the antigenic component comprises an attenuated recombinant viral vector, a naturally or genetically engineered attenuated live virus or microorganism, an inactivated virus or microorganism, a coccidial microorganism, a protein subunit, a unicellular parasite, a multicellular parasite or any combination of the foregoing.

41. The immunization or vaccine composition of claim 40 wherein the Coccidia microorganism is a Coccidia precocious microorganism.

42. An immune or vaccine composition according to claim 37, wherein the antigenic component comprises: an Eimeria sp or antigen thereof, an Escherichia coli (e.coli) or antigen thereof, a Mycoplasma hyopneumoniae (m.hyo), a Bovine Diarrhea Virus (BDV) antigen, a recombinant canarypox vector containing and capable of expressing at least one protective immunogen in vivo, an inactivated full length rabies glycoprotein, a erysiphe sp, a erysiphe rhusiopathiae, a surface protective antigen from erysiphe rhusiopathiae (SpaA), a surface protective antigen (SpaA) fusion protein comprising at least a portion of at least one additional immunogen, a Clostridium perfringens (Clostridium perfringens) B/C toxin, a Clostridium perfringens (Clostridium) toxin, a Clostridium toxin (Clostridium) toxin, a Clostridium perfringens (Clostridium toxin, clostridium tetanii) B/C toxin, or a combination thereof.

43. The immunization or vaccine composition according to claim 42 wherein the surface protective antigen (SpaA) fusion protein comprising at least a portion of at least one additional immunogen is a SpaA-FlaB fusion protein or a SpaA-FlaB-His fusion protein.

44. An immunising or vaccine composition according to claim 42, wherein the antigenic component comprises or consists of inactivated full length rabies glycoprotein.

45. The immunization or vaccine composition according to claim 42 wherein the antigenic component comprises or consists of a Clostridium perfringens B/C toxin, a Clostridium perfringens D toxin, a Clostridium septicum toxin, a Clostridium novyi toxin, a Clostridium tetani toxin, or a combination thereof.

46. An immune or vaccine composition according to claim 42, wherein the antigenic component comprises Clostridium perfringens B/C toxin, clostridium perfringens D toxin, clostridium septicum toxin, clostridium nokohlii toxin and Clostridium tetani toxin.

47. An immunising or vaccine composition according to claim 42, wherein the antigenic component comprises a surface protective antigen (SpaA).

48. An immunisation or vaccine composition according to claim 42, wherein said antigenic component includes a fusion protein including a surface protective antigen (SpaA).

49. An immunisation or vaccine composition according to claim 42, wherein said antigenic component comprises an attenuated fowlpox virus or a DNA plasmid which contains and is capable of expressing an influenza gene in vivo.

50. The immunization or vaccine composition according to claim 42 wherein the antigenic component comprises an attenuated fowlpox virus or a DNA plasmid containing and capable of expressing the rabies glycoprotein gene in vivo.

51. Use of a vaccine composition according to claim 42 in the manufacture of a medicament for the treatment of bovine against infection by bacteria.

52. Use of a vaccine composition according to claim 46 in the manufacture of a medicament for treating a canine or equine canine against infection caused by influenza.

53. Use of a vaccine composition according to claim 46 in the manufacture of a medicament for the treatment of canines against infection by rabies virus.

54. An avian coccidiosis vaccine for in ovo administration, said vaccine comprising:

(a) Vaccine adjuvant which is a pharmaceutically acceptable salt of a linear or branched polyacrylic acid polymer, characterized in that said polyacrylic acid polymer salt has a weight average molecular weight Mw in the range of 350-650kDa and a polydispersity index of less than or equal to 4; and comprises less than 0.005% w/w of an oxidizing agent, based on the total dry weight of the polyacrylic acid polymer salt, and wherein the polyacrylic acid polymer consists entirely of acrylic acid units; and

(b) A protozoan antigen selected from the group consisting of: (1) one or more recombinantly expressed proteins; (2) One or more proteins or other macromolecules isolated from the protozoa by conventional means; (3) A whole cell extract or preparation from said protozoa; and (4) inactivated, live or live precocious coccidia selected from the group consisting of: eimeria acervulina (Eimeria acervulina), eimeria adenoviridae (Eimeria adeneides), eimeria brunetti (Eimeria brunetti), eimeria colchica (Eimeria colchici), eimeria curvata (Eimeria curvata), eimeria dispersa (Eimeria disperisa), eimeria duodenale (Eimeria duodenalis), eimeria fitzei (Eimeria fratercula), eimeria dubia curvata (Eimeria fratercula), eimeria gawariana (Eimeria volvularia), avirulent Eimeria (Eimeria innocula), eimeria praecox (Eimeria praecox), eimeria maxima (Eimeria maxima), eimeria zukii (Eimeria melegadis), eimeria both Eimeria and Eimeria mitis (Eimeria meleagris), and Eimeria mitis (Eimeria mitis), eimeria necatrix (Eimeria necatrix), eimeria faberi (Eimeria phasiani), eimeria protoceri (Eimeria procera), eimeria avicularis (Eimeria tenella), and combinations thereof.

55. Use of a vaccine composition according to claim 42 in the manufacture of a medicament for the treatment of infection in bovines against E.coli or Mycoplasma hyopneumoniae, wherein the antigenic component comprises E.coli or Mycoplasma hyopneumoniae.

56. An immunisation or vaccine composition according to claim 42, wherein said antigenic component includes Mycoplasma hyopneumoniae.

57. Use of a vaccine composition according to claim 56 in the manufacture of a medicament for the treatment of infection in a porcine animal by Mycoplasma hyopneumoniae.

58. An immunising or vaccine composition according to claim 42, wherein the antigenic component comprises FIV.

59. Use of a vaccine composition according to claim 58 in the manufacture of a medicament for the treatment of an infection in a feline resulting from FIV.

60. The vaccine composition according to claim 42, wherein the antigenic component comprises a cancer antigen.

61. Use of a vaccine composition according to claim 60 in the manufacture of a medicament for treating a subject against cancer.

62. An immunising or vaccine composition according to claim 42, wherein the antigenic component comprises Canine Coronavirus (CCV).

63. Use of a vaccine composition according to claim 62 in the manufacture of a medicament for treating canines against infection by Canine Coronavirus (CCV).

64. An immune or vaccine composition according to claim 42 wherein the antigenic component comprises bovine rotavirus.

65. Use of a vaccine composition according to claim 64 in the manufacture of a medicament for the treatment of bovine animals against infection by bovine rotavirus.

66. The immunization or vaccine composition according to claim 42 wherein the antigenic component comprises Canine Influenza Virus (CIV).

67. Use of a vaccine composition according to claim 66 in the manufacture of a medicament for the treatment of canines against infection by Canine Influenza Virus (CIV).